Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli

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Goméz-Quintero, Melissa D. Morales-Moreno, Erick G. Valdés-Galindo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535821/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The CRISPR-Cas12a system is a groundbreaking tool that has seen an ample use for genome editing and diagnostics in biotechnology and biomedicine research labs. Despite its increasing use, there is a lack of studies on optimizing Cas12a protein production at lab-scale using straightforward protocols. This study aimed on enhancing the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli . Through careful adjustments of simple parameters, the production of AsCas12a was remarkably increased. Optimized conditions involved using the BL21(DE3) strain, TB medium with 1% glucose, induction with 0.3 mM IPTG for at least 6–9 h and incubation at 30°C. Notably, these conditions deviate from conventional production protocols for Cas12a and related proteins such as Cas9 from Streptococcus pyogenes . Upon combination of all optimized conditions bacterial production of AsCas12a improved ~ 3 times, passing from 0.95 mg / mL of bacterial lysate volume, for non-optimized conditions, to 3.73 mg/mL in the optimal ones. The production yield of AsCas12a protein, after chromatographical purification increased ~ 4.5 times, from 5.2 to 23.4 mg/L (culture volume) without compromising its functionality at all. The purified AsCas12a protein retained full activity for programmable in vitro DNA cis -cleavage and for collateral trans -activity, which was used to detect the N gene from SARS-CoV-2. This optimized method offers an efficient and high-yield AsCas12a protein production using materials and conditions that are accessible to many research labs around the world. CRISPR-Cas12a Protein biosynthesis Recombinant production Optimization Lab-scale production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Points Lab-scale production of Cas12a was highly boosted using a simple strategy. Optimized conditions increased AsCas12a production yield up to 4.5 times. Method offers efficient, straightforward and high-yield Cas12a production. Introduction The CRISPR-Cas12a system, formerly known as Cpf1, stands as a potent molecular tool with diverse applications in genome editing, molecular diagnostics, and various biotechnological and biomedical fields (Paul and Montoya 2020 ; Shi et al. 2021 ; Khan and Sallard 2023 ). Given the manifold applications of the CRISPR-Cas12a system, there exists a widespread interest in the small-scale, high-yield biosynthesis of the Cas12a protein among research laboratories in both developed and non-developed countries (Bandyopadhyay et al. 2020 ; Jirawannaporn et al. 2022 ; Han et al. 2022 ; Paenkaew et al. 2023 ). Many laboratories around the world need a continuous supply of the protein to carry on and continue with their research activities. Although Cas12a protein is available commercially, its high cost makes research labs to prefer to produce and purify themselves the protein. However, the current available lab protocols are low efficiency and have not been optimized. Despite the increasing application of Cas12a, there remains a noticeable lack of studies focused on its small lab-scale production without the need to use a bioreactor or specialized bacterial strains. While some groups have reported the recombinant production of Francisella novicida Cas12a protein (Zetsche et al. 2015 ; Dong et al. 2016 ; Strohkendl et al. 2018a ; Chen et al. 2018 ), only very few reports (Mohanraju et al. 2018 ; Martin et al. 2023 ) have described detailed protocols for the biosynthesis of other relevant orthologues such as Acidaminococcus sp Cas12a protein (AsCas12a). Surprisingly, there is a notable gap in the literature regarding the systematic study of production of Cas12a, underscoring the need for comprehensive investigations in this area. Consequently, an exploration of the recombinant production of Cas12a proteins is imperative to advance CRISPR-Cas12a-based research, with a particular focus on the AsCas12a variant in this study. The endonuclease AsCas12a is classified as a type V CRISPR-Cas system, as it serves as the sole effector protein responsible for DNA cleavage. Cas12a possesses key attributes that position it as a robust alternative to the widely utilized CRISPR-Cas9 system (Swarts and Jinek 2018 ). Notably, the crRNA of Cas12a is shorter than Cas9 gRNA (40–44 vs 100 nt), and it cleaves dsDNA in a staggered manner. Cas12a exhibits a higher precision in binding to dsDNA sequences compared to Cas9, boasting a substantial affinity ( K D ~fM) and displays a pronounced trans -cleavage activity upon ssDNA (commonly referred to as collateral activity) (Jinek et al. 2012 ; Zetsche et al. 2015 ; Dong et al. 2016 ; Gao et al. 2016 ). These distinctive features make the Cas12a protein an excellent choice within the CRISPR-Cas systems for both fundamental and applied research. AsCas12a forms a ribonucleoprotein complex (RNP) in conjunction with a CRISPR RNA (crRNA, also known as guide RNA or gRNA). The RNP makes a highly specific cleavage of both DNA strands upon binding the target DNA with a remarkable affinity ( K D of 54 fM) (Zetsche et al. 2015 ; Strohkendl et al. 2018a ). Particularly effective with T-rich genomes, AsCas12a recognizes the PAM 5’ TTTV 3’ (Kim et al. 2017 ). Comprising 1,307 amino acid residues and a molecular weight of 151.2 kDa, the AsCas12a is structured into REC and NUC lobes (Dong et al. 2016 ; Gao et al. 2016 ). The NUC lobe contains the RuvC-I, II and III domains, responsible for the ultimate cleavage of dsDNA. Distinct from SpyCas9, AsCas12a exhibits unique features (Swarts and Jinek 2018 ). AsCas12a uses a shorter guide RNA, lacks a tracrRNA, cleaves dsDNA with 5’ overhangs and has a longer distance between the seed sequence and the cleavage site (Swarts and Jinek 2018 ). Despite these differences, AsCas12a robustly and efficiently cleaves dsDNA in human cells (Zetsche et al. 2015 ), displaying on-target efficiencies comparable to the widely used SpyCas9 (Kleinstiver et al. 2016 ). All this underscores the need of studies focused on optimizing the recombinant production of AsCas12a in E. coli , the most commonly employed heterologous expression system of choice for many research labs around the world (Rosano and Ceccarelli 2014 ). This study presents a systematic study aimed to identify optimal conditions for the lab-scale production of recombinant AsCas12a in E. coli as expression system, comparing them with initial conditions based on standard available protocols (Mohanraju et al. 2018 ; Martin et al. 2023 ) (Fig. 1 ). It was studied the effect the influence of several parameters, including E. coli strain, IPTG concentration, culture media, media supplementation with glucose, and post-induction temperature (Fig. 1 A). The protein expression was monitored under various conditions, and the most effective combinations were identified to ensure efficient protein expression. The significance of studies that report improved conditions to express and purify CRISPR-Cas proteins is of large relevance, as they contribute to advancing the entire CRISPR-Cas field by facilitating the availability of proteins for experimentation. It was considered that the optimized conditions for the heterologous production of AsCas12a protein in E. coli will prove beneficial to research laboratories engaged in CRISPR-based studies that have available basic equipment and materials for heterologous bacterial expression. Materials and Methods Plasmids The pMBP-AsCas12a plasmid was employed for the production of AsCas12a protein (Fig. 1 ), generously provided by Prof. Ilya Finkelstein at the University of Texas-Austin (Addgene #113430) (Fig. 1 B). This plasmid harbors an ampicillin resistance gene and the AsCas12a gene, featuring a His10-tag, all under the control of a T7 promoter. The plasmid pPET28a-SARS-CoV-2-geneN was utilized for cis and trans cleavage assays of the AsCas12a protein, a generous gift from Dr. Luis Brieba de Castro from LANGEBIO-UGA, Mexico, containing the N gene from the SARS-CoV-2 Wuhan reference genome. Materials The Luria Bertani (LB) Broth culture medium was purchased from Invitrogen (Waltham, Massachusetts, USA). The highly enriched Terrific Broth (TB) medium was prepared with 24 g/L yeast extract (Formedium, Hunstanton, UK), 12 g/L Tryptone (Sigma-Aldrich, St. Louis, Missouri, USA), 4 mL glycerol (JT Baker, Phillipsburg, NJ, USA), and 0.17 M phosphate buffer (pH 7) (K 2 HPO 4 and KH 2 PO 4 , Sigma-Aldrich). Glucose, ampicillin, kanamycin, 2-Mercaptoethanol and bromophenol blue were purchased from Sigma-Aldrich. Isopropil ß-D-1-thiogalactopyranoside (IPTG) was obtained from Invitrogen (Carlsbad, USA). Agar, acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate, and glycine were purchased from Biorad (Hercules, California, United States). N, N, N′, N′-Tetramethyl ethylenediamine (TEMED) was acquired from IBI Scientific (Dubuque, Iowa, United States), while Tris was obtained from Gold Bio (St. Louis, Missouri, United States). Furthermore, gRNAs and ssDNA reporter probe were synthesized by Integrated DNA Technologies-IDT (Coralville, USA). Bacterial strains and culture media preparation The strains employed in the assays for recombinant protein biosynthesis, BL21 (DE3), Rosetta (DE3), BL21(pLysS), Tuner ™ (DE3), and NiCo (DE3), were generously provided by Dr. Patricia Cano Sánchez and Dr. Corina-Diana Ceapă from the Institute of Chemistry, UNAM, Mexico. The LB broth was prepared by dissolving 12.5 g of LB media powder in 1 L of distilled water. TB solution was prepared as mentioned above. These media solutions were sterilized using an autoclave (Sterilite 24, Fisher Scientific, Waltham, USA). As needed, LB and TB media were enriched by supplementing with glucose, reaching a final concentration of 1% (w/v), previously sterilized using 0.2 µm filters (Sartorius, Germany). Bacterial transformation and plasmid production Bacterial transformation was achieved through the thermal shock method. Competent E. coli DH5α (or any other strain) were placed on ice for 5 min with 2 µL plasmid. The mixture underwent a temperature shift to 42°C for 30 s, followed by an incubation on ice for 5 min. The sample then was mixed with 100 µL LB and incubated at 37°C for 1 h. The mixture was added to agar plates supplemented with 100 µg/mL ampicillin and incubated overnight at 37°C. A colony of bacteria transformed with the plasmid was grown overnight in 5 mL LB medium, incorporating the necessary antibiotic (ampicillin or kanamycin) at 100 µg/mL. Subsequently, plasmids were purified from the culture using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, California, USA). Expression of protein AsCas12a The initial experimental conditions were based on previously published protocols for the expression of Cas12a (Mohanraju et al. 2018 ; Strohkendl et al. 2018a ) (Fig. 1 C). To initiate the pre-inoculum, a colony was selected from an agar plate and cultivated in a 250 mL Erlenmeyer flask containing 100 mL media (LB or TB, depending on the assay) supplemented with 100 µg/mL of ampicillin. The pre-inoculum was incubated at 37°C for 16 h and 225 rpm using a MaxQ 4,000 shaker incubator equipped with controlled temperature settings (Thermo Scientific, Waltham, Massachusetts). Following this, the pre-inoculum was diluted to an optical density (OD) of 0.1 in 100 mL of culture media (TB or LB, with or without 1% glucose, incorporating 100 µg/mL of ampicillin). The culture was then incubated at 37°C with 225 rpm until the OD reached a value of 0.6–0.8 (approximately 1.5 h). OD was measured in a BioPhotometer plus spectrophotometer (Eppendorf, Hamburg, Germany). Then, the culture was incubated at the desired experimental temperature (12, 18, 30 or 37°C) with 225 rpm. After 30 min, IPTG induction was carried out (0.3, 0.6, 1, 1.3, 1.6 mM), and the culture was incubated for the designated time. Cell lysis and sample preparation Bacterial samples taken during the growth curves were treated in the following way before their analysis of protein expression. 1 mL of bacterial culture was centrifuged at 4°C for 5 min at 13,000 rpm using a microcentrifuge AccuSpin Micro 17R (Fisher Scientific, Waltham, USA). The supernatant was discarded, and the pellet was resuspended in 100 µL cell lysis buffer [200 mM 2-Mercaptoethanol, 25% glycerol, 10% SDS, 0.5 M Tris-HCl (pH 6.8), and 0.5% bromophenol blue] and heated up to 95°C for 5 min using an Isotemp Thermoblock (Fisher Scientific, Waltham, USA). Finally, the solution was centrifuged at 4°C for 5 min at 13,000 rpm and the supernatant was recovered and stored at -20°C for further analysis, then only the soluble fraction was further analyzed. Cell lysis buffer without bromophenol blue was used when total protein concentration present in the bacteria culture was measured by Nanodrop. When very dense cultures were lysed, they were centrifugated during 55 min to eliminate the excess of debris. Quantification of total soluble protein The total protein quantification was performed using a NanoDrop One UV-Vis spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and by micro-bicinchoninic acid assay (BCA) using the Pierce™ BCA Protein Assay Kit (ThermoScientific). For NanoDrop, approximately 1.5 µL of the cell lysate were scanned using the protein A280 program after a buffer baseline correction determined by the device. For micro–BCA microplate Assay 150 µL of sample (2 µL of bacterial lysate diluted to a volume of 500 µL) were loaded into a 96-well microplate by triplicate together with the albumin standard (prepared following the instructions of the protocol). Then, 150 µL of the BCA working reagent was added to each well and the mixture was incubated for 2h at 37°C to finally read in a Cytation 5 Multi Mode Reader (BioTek, Santa Clara, USA) at 562 nm. The obtained results were analyzed and plotted using GraphPad 10. SDS-PAGE analysis Protein expression from lysed samples was analyzed by electrophoresis in SDS-PAGE gel using a 247.6 mM Tris-glycine (pH 8) as running buffer at 160 V for 45 min. Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standard was used as a molecular weight marker (Bio Rad, Hercules, California, United States). Gels were water-washed three times and then stained with SimplyBlue™ Safe Stain (Thermo Fisher, Waltham, USA). Gels were imaged in an Azure 200 Gel Imager (Azure Biosystems, California, USA). Densitometric analysis was carried out using ImageJ software. The percentage of AsCas12a protein present in each sample was determined by measuring the intensity of the band corresponding to the AsCas12a protein band relative to the total protein band intensity present in the lane (Supplementary Fig. S1 ). Chromatography protein purification Protein purification was performed following a previously reported purification protocol (Morales-Moreno et al. 2023 ). The lysed product from 1 L was purified in an ӒKTA pure™ chromatography system using affinity, ionic exchange, and molecular size exclusion chromatography. Metal affinity chromatography was done with a HisTrapTM HP 5 mL column, and the protein was eluted with 250 mM imidazole at a flow rate of 2 mL/min The Histidine tag was cleaved off using TEV protease during an overnight dialysis at 4°C. The ionic exchange chromatography was carried out using a HiTrap Heparin HP 5 mL column and the protein was eluted with IEx-B buffer (2M potassium chloride) at a flow rate of 2 mL/min. Finally, the molecular size exclusion chromatography was performed using a HiLoad 16/600 Superdex 200 pg 125 mL column and at a buffer flow rate of 0.5 mL/min. Cis-cleavage activity on pDNA AsCas12a:crRNA ribonucleoprotein complexes (RNPs) were assembled by incubating for 30 min at 37°C a 1:1 molar mixture of the purified AsCas12a protein and crRNA designed towards the N gene (Morales-Moreno et al. 2023 ) of SARS-CoV-2 in 1X NEB 2.1 buffer (New England Biolabs, Ipswich, Massachusetts, USA). Then, RNP complexes were mixed with the target dsDNA (pPET28a-SARS-CoV-2-geneN) to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively (molar proportion RNP:pDNA of 10:1) and incubated in a thermoblock at 37°C during 1 h. Then, samples were mixed with Gel Loading Dye 6X (Invitrogen) and evaluated in an 1% agarose gel (Thermo Fisher) using TAE 1X as running buffer (Thermo Fisher) at 140 V for 45 min after staining with SYBR™ Safe DNA Gel Stain (Invitrogen). Gels were imaged with an Azure 300 gel imager system (Azure Biosystems, Dublin, CA). Trans-cleavage activity on ssDNA Trans -cleavage activity assay was evaluated following a previous reported protocol (Morales-Moreno et al. 2023 ). In short, RNPs were assembled as previously indicated and mixed with the target pPET28a-SARS-CoV-2-geneN to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively in 1X NEB 2.1 buffer. As a reporter probe 250 nM of ssDNA attached to FAM and a quencher was used. Trans -cleavage activity on ssDNA was followed by fluorescence at 37°C during 1 h using a Cytation 5 Cell Imaging Multi Mode Reader (BioTek, Santa Clara, USA). Statistical evaluations Ordinary one-way ANOVA tests were conducted using the GraphPad Prism 10 software on all the SDS-PAGE densitometry assays results of the supernatants obtained from the lysed bacteria of the cultures (IPTG concentration, Temperature, Culture Media, Glucose supplementation and E. coli Strain) to compare if there was a significant difference between the original parameters and the new experimental parameters. Values of P greater than 0.5 were considered as non-significant. Results Effect of IPTG concentration First, it was explored a 0.3 to 1.6 mM of IPTG concentrations over a 24 h induction period (Fig. 2 ). The level of AsCas12a protein production upon IPTG concentration was followed by analyzing cell lysates in SDS-PAGE. The SDS-PAGE illustrated successful induction of AsCas12a at all tested IPTG levels (Fig. 2 a, Supplementary Fig. S2). Densitometric analysis revealed a decreasing trend in the total AsCas12a protein band percentage relative to the total band intensity with increasing IPTG concentrations (21.1, 19.9, 18.9, 20.3, and 17.1% for 0.3, 0.6, 1, 1.3, and 1.6 mM IPTG, respectively) (Fig. 2 b). However, the differences were not statistically significant between them. Effect of temperature on AsCas12a expression To assess the impact of temperature on protein expression, recombinant E. coli cultures induced with 0.3 mM IPTG were incubated at various temperatures. After 9 h of induction, cell lysates were analyzed by SDS-PAGE to observe the percentage of produced AsCas12a protein (Fig. 3 , Supplementary Fig. S3). AsCas12a protein expression was observed across all temperatures, albeit in varying amounts (Fig. 3 a). Densitometric analysis revealed that the highest production of AsCas12a proteins was approximately 27% (of the total soluble protein) at 30°C (Fig. 3 b). This contrasts with 12.2% (12°C), 17.9% (18°C), and 13.7% (37°C). The percentage of protein at 30°C represents a ~ 2.2-fold and ~ 1.5-fold increase compared to 12°C and 18°C, respectively. Effect of culture media We investigated the impact of highly accessible media, such as LB and TB, for AsCas12a production, and assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni 2019 ). After inducing AsCas12a expression with 0.3 mM IPTG and incubating 30°C for 9 h, the percentage of AsCas12a in the total soluble protein (present in cell lysate) was measured with SDS-PAGE (Fig. 4 , Supplementary Fig. S4). After comparing LB and TB, it was observed a ~ 1.6-fold improvement (16.9 and 27.2%, respectively). However, supplementing TB with 1% glucose increased AsCas12a protein production from 27.2 to 31.5%, whereas LB + 1% glucose reduced from 16.9 to 15.9% (Fig. 4 a). Changing LB to TB + 1% glucose represented a ~ 1.9-fold increment. Notably, TB + 1% glucose production was high enough that the lysed sample caused streaks on SDS-PAGE bands. Effect of strain Employing five commonly used E. coli DE3 strains (BL21, Tuner, Rosetta, PlysS, and NiCo), we aimed to identify the most efficient one for AsCas12a biosynthesis under our previously optimized conditions (TB + 1% glucose, 0.3 mM IPTG, and 30°C). After analyzing cell lysates with SDS-PAGE, it was revealed distinct protein production levels among the strains (Fig. 5 , Supplementary Fig. S5). BL21 exhibited prominent production, followed by Rosetta and NiCo, while pLysS and Tuner showed the lowest (Fig. 5 a). Anomalous electrophoretic mobility was observed in bands with high protein expression, notably in BL21 and Rosetta. The AsCas12a protein production was quantified (Fig. 5 b). The quantitative analysis confirmed BL21 as the top performer, 31.5% from the total soluble protein corresponded to AsCas12a protein, followed by Rosetta with 26.7%, NiCo 18.2%, Tuner 15.5%, and pLysS with 14.0%. Comparison between initial and improved conditions Next, it was assessed with detail the impact of optimized conditions (BL21(DE3) strain, TB + 1% glucose, 0.3 mM IPTG, and 30°C) and compared against initial non-optimized conditions (BL21(DE3) strain, LB, 1 mM IPTG, and 12°C). This was done by following over 24 h (post-IPTG induction) cell growth kinetics, production of total soluble protein, and production of AsCas12a protein in both conditions (Fig. 6 ). After 9h, optimized conditions exhibited significantly faster bacterial growth, reaching a maximum OD of 6.6, whereas non-optimized conditions exhibited an OD of 2.1 absorbance units. It was only after 23h that the non-optimized conditions reached an OD of 2.7 (Fig. 6 a). A similar trend was observed for amount of total soluble protein (determined by BCA). After ~ 9h the optimized conditions reached ~ 15 mg/mL (relative to volume of bacterial lysate), whereas for non-optimized conditions was ~ 10 mg/mL (Fig. 6 b). Even after 24 h, protein production in non-optimized conditions was still lower (~ 11.5 mg/mL). To correlate the previous results of production of total soluble protein with AsCas12a protein production, SDS-PAGE and densitometric analysis were carried out (Fig. 6 C-E, Supplementary Fig. S6). The quantitative analysis of SDS-PAGE (Fig. 6 c) confirmed that ~ 25% of the total soluble protein produced at 9 h corresponded to AsCas12a protein, remaining constant thereafter (Fig. 6 d). In contrast, non-optimized conditions showed a slower increase, reaching ~ 13% of AsCas12a protein at 9h and ~ 16% only until 24 h. All this means that the concentration of AsCas12a protein produced at 9 h for optimized conditions was ~ 3.5 mg/mL (relative to volume of cell lysate), and ~ 1.2 mg/mL for non-optimized conditions, representing a ~ 3-fold increment (Fig. 6 e). A summary of all values is presented in Table 1 . Table 1 Summary of the results obtained between optimized and non-optimized conditions in bacterial lysates. Optical Density (A.U.) a Total Soluble Protein (mg/mL) b AsCas12a (%) c AsCas12a (mg/mL) d Time 9 h 24 h 9 h 24 h 9 h 24 h 9 h 24 h Non-optimized conditions 2.1 2.6 11 11.2 13.2 16 1.2 1.8 Optimized conditions 6.6 7.1 15 15 24.8 23.5 3.1 3.5 a Bacterial growth measured as optical density. b Total amount of cleared soluble protein per volume of bacterial lysate determined by BCA. c Percentage of protein AsCas12a in total soluble protein determined by densitometry in SDS-PAGE. d Total amount of protein AsCas12a produced in the bacterial lysate determined from SDS-PAGE and BCA. Purification and functional evaluation of AsCas12a protein To assess the functionality of the recombinant AsCas12a produced under optimized conditions, first the protein AsCas12a was purified and then we examined its cis - and trans -cleavage activities (Fig. 7 ). The protein was expressed under optimized and non-optimized conditions in 1 L flasks, purified with Ni-NTA affinity, ionic exchange, and molecular size exclusion chromatography (Morales-Moreno et al. 2023 ), and characterized by SDS-PAGE (Fig. 7 a). After purification the production yield of AsCas12a protein was 23.4 mg/L (of bacterial culture), whereas for non-optimized conditions was 5.2 mg/L. Western blot confirmed its identity and successful purification (Supplementary Fig. S7). Then, it was tested the functional performance of the AsCas12a protein to cleave dsDNA, cis -activity, (Fig. 7 b). The cis -cleavage activity of RNP complex (AsCas12a: crRNA) efficiently digested the supercoiled target pDNA (7,253 kbp), while leaving intact the non-target pDNA (9,210 kbp). Finally, it was evaluated the trans -cleavage activity on ssDNA, also called collateral activity (Fig. 7 c). It was used two gRNAs that recognize the sequence of the nucleocapsid gene of SARS-CoV-2 located in a pDNA. To evaluate the functional performance of AsCas12a protein, it was followed the fluorescence increase upon cleavage of a FAM-quencher labeled reporter ssDNA after recognition of the target pDNA sequence. The correct trans -cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time. Discussion CRISPR-Cas systems have become one of the most relevant biomolecular systems for current biotechnological research in many labs around the world. Warrantying a continuous and cheap supply is enormously important for doing basic research and technology development in a productive and timely manner. The system CRISPR-Cas12a is the second most used just below CRISR-Cas9 (Paul and Montoya 2020 ). Besides genetic engineering, Cas12a has ample applications in biosensor development (Shi et al. 2021 ; Hernandez-Garcia et al. 2022 ). In particular, AsCas12a is one of the most used between the various homologous of Cas12a. AsCas12a protein is commonly produced in Escherichia coli ; however, an efficient production of the protein has not been reported. Having an optimized production of protein AsCas12a at lab scale would benefit many research labs that continuously use it. Furthermore, optimal production of protein AsCas12a has more chances of being adopted if is based on materials that are easily accessible and available to most research labs, also, if the optimization is based on simple parameters. That is why here we investigated the effect of multiple simple parameters that are important for the efficient production of AsCas12a protein. First, we explored the effect of 0.3 to 1.6 mM of IPTG concentration over a 24 h induction period (Fig. 2 ). Our results demonstrated that IPTG concentrations above 0.3 mM had not significant impact. This suggests that 0.3 mM concentration is enough to effectively induce AsCas12a expression in E. coli . This IPTG concentration is very close to previously reported values of 0.13 and 0.2 mM IPTG (Jinek et al. 2012 ; Mohanraju et al. 2018 ; Chen et al. 2018 ; Martin et al. 2023 ); but contrasts with the 1 mM used by others (Strohkendl et al. 2018b ). Next, we investigated the effect of temperature on AsCas12a expression. After assessing the impact of various temperatures (12, 18, 30 and 37ºC) on protein expression in recombinant E. coli cultures induced with 0.3 mM IPTG (Fig. 3 ), we found that 30°C was the best post-induction temperature for successful enhancement of protein production. Producing at 30°C increased ~ 2.2-fold and ~ 1.5-fold the production of AsCas12a protein compared to 12°C and 18°C, respectively. This finding is significant since most of the protocols reported for producing AsCas12a using E. coli commonly used temperatures of 12 or 18ºC but not 30ºC (Jinek et al. 2012 ; Mohanraju et al. 2018 ; Chen et al. 2018 ; Martin et al. 2023 ). Other aspect of interest in our study was to evaluate the effect of culture media. Analyzing effect of culture media is crucial for optimizing recombinant protein expression (Rosano and Ceccarelli 2014 ). For that reason, we investigated the impact of highly available media, such as LB and TB, for AsCas12a production (Fig. 4 ). Also, we assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni 2019 ). TB supplemented with 1% glucose resulted advantageous for recombinant production of protein AsCas12a, even surpassing LB, LB + 1% glucose, and TB alone in protein production. The impact on production of adding glucose to TB was very relevant. This positive effect may stem from the role of glucose as an alternative carbon source to an already rich carbon source media (TB contains glycerol), accelerating total biomass and protein production (Choi et al. 2006 ). Glycerol in TB also aids in preventing acetate formation and serves as an alternative carbon source. Conversely, the negative effect of glucose in LB may be attributed to its transformation into acetates, hindering growth, inhibiting protein formation, and diverting carbon away from biomass to protein product (Wong et al. 2008 ). The final parameter was the bacterial strain. The choice of E. coli strain may significantly influence the recombinant production of protein AsCas12a since it has been observed that E. coli strains play important roles for heterologous protein production (Zhang et al. 2022 ; Pouresmaeil and Azizi-Dargahlou 2023 ). Among all the studied strains, BL21(DE3) resulted the most efficient (Fig. 5 ). This is positive judging that is a very common strain in many biotechnological research labs. On the other hand, it could be interesting to explore new E. coli strains that are being developed for efficient heterologous production of proteins under the strategies of synthetic biology (Zarzhitsky et al. 2020 ; Yang et al. 2022 ; Zhang et al. 2022 ). Finally, we compared between initial and improved conditions. We assessed in detail the impact of combining our best-found conditions against initial non-optimized conditions which were based of typical reported protocols for Cas12a protein production (Martin et al., 2023 ; Mohanraju et al., 2018 ; Strohkendl et al., 2018a ). The higher values obtained for OD and concentration of total soluble protein and total AsCas12a protein can tell us that our optimization strategy was successful (Fig. 6 ). Of particular relevance is the production of AsCas12a. The concentration of AsCas12a protein produced at 9 h for optimized conditions was ~ 3.5 mg/mL (relative to volume of cell lysate), which contrast with ~ 1.2 mg/mL obtained with non-optimized conditions. The difference represents a ~ 3-fold increment (Fig. 6 e). The striking differences between both conditions could be explained by the usage of the rich media TB + 1% glucose and a higher incubation temperature for the optimized conditions. The efficiency of optimized conditions became evident, indicating their suitability for high-level AsCas12a protein production. In fact, the non-optimized conditions will require much longer than 24 h to produce similar levels of protein. All this demonstrates that our optimized conditions are producing more protein in shorter time. Verifying the functional performance of the produced and purified AsCas12a protein is of outmost importance. We could verify the cleavage of dsDNA (Fig. 7 b), cis -activity, which one of the earliest and most common application of AsCas12a (Zetsche et al. 2015 ; Kleinstiver et al. 2016 ; Bandyopadhyay et al. 2020 ; Han et al. 2022 ; Yang et al. 2023 ). Also, the evaluation of the trans -cleavage activity on ssDNA, also called collateral activity, is crucial since it is the base of multiple methods for genetic detection and clinical diagnostics in labs around the world (Chen et al. 2018 ; Lee Yu et al. 2021 ; Jirawannaporn et al. 2022 ; Morales-Moreno et al. 2023 ; Yang et al. 2023 ; Paenkaew et al. 2023 ). The trans -cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time (Fig. 7 c), showcasing the protein full collateral activity and ability to detect specific DNA sequences. Both activities demonstrated to work between acceptable standards. In conclusion, this study identified several parameters that significantly enhanced the heterologous lab-scale production of AsCas12a protein in recombinant E. coli cultures. Optimized conditions were found for all the studied parameters (IPTG concentration, induction temperature, culture media, and type of bacterial strain). Combination of all optimized conditions increased ~ 3 times the bacterial production of protein AsCas12a in a shorter time (~ 9h). The protein production yield after purification was 23.4 mg/L of culture volume, representing a 4.5-fold increase in comparison to the non-optimized conditions. Importantly, the purified AsCas12a protein retained full cis and trans cleavage activities, which are essential for various downstream CRISPR-Cas applications. These optimized protein production conditions offer a valuable resource for research labs seeking higher yields of functional AsCas12a protein, all in a more efficient timeframe and using a simple protocol that requires accessible materials and equipment. Declarations Funding: This work was supported by UNAM-PAPIIT (IN210121 & IV200820). The authors gratefully acknowledge support from Agencia Mexicana de Cooperacion Internacional para el Desarrollo (AMEXCID)—Secretaria de Relaciones Exteriores Mexico (Proyectos COVID-19). Conflicts of interest/Competing interests: The authors declare no conflict of interest for this manuscript. Data availability (data transparency): Supplementary information is included. Code availability: Not applicable. Authors' contributions OSQG and AHG conceived and designed research. OSQG, MDMM, EGVG and RECG conducted experiments. OSQG and AHG analyzed data. AHG wrote the manuscript with input from OSQG. All authors read and approved the manuscript. References Bandyopadhyay A, Kancharla N, Javalkote VS, Dasgupta S, Brutnell TP (2020) CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement. Front Plant Sci 11. https://doi.org/10.3389/fpls.2020.584151 Carmignotto GP, Azzoni AR (2019) On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains. J Biotechnol 306:62–70. https://doi.org/10.1016/j.jbiotec.2019.09.012 Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (80- ) 360:436–439. https://doi.org/10.1126/science.aar6245 Choi JH, Keum KC, Lee SY (2006) Production of recombinant proteins by high cell density culture of Escherichia coli . Chem Eng Sci 61:876–885. https://doi.org/10.1016/j.ces.2005.03.031 Dong D, Ren K, Qiu X, Zheng J, Guo M, Guan X, Liu H, Li N, Zhang B, Yang D, Ma C, Wang S, Wu D, Ma Y, Fan S, Wang J, Gao N, Huang Z (2016) The crystal structure of Cpf1 in complex with CRISPR RNA. Nature 532:522–526. https://doi.org/10.1038/nature17944 Gao P, Yang H, Rajashankar KR, Huang Z, Patel DJ (2016) Type v CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Res 26:901–913. https://doi.org/10.1038/cr.2016.88 Han X, Yang Y, Han X, Ryner JT, Ahmed EAH, Qi Y, Zhong G, Song G (2022) CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry. Plant Cell, Tissue Organ Cult 148:217–229. https://doi.org/10.1007/s11240-021-02177-1 Hernandez-Garcia A, Morales-Moreno MD, Valdés-Galindo EG, Jimenez-Nieto EP, Quezada A (2022) Diagnostics of COVID-19 Based on CRISPR–Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update. Diagnostics 12:1434. https://doi.org/10.3390/diagnostics12061434 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (80- ) 337:816–821. https://doi.org/10.1126/science.1225829 Jirawannaporn S, Limothai U, Tachaboon S, Dinhuzen J, Kiatamornrak P, Chaisuriyong W, Bhumitrakul J, Mayuramart O, Payungporn S, Srisawat N (2022) Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32. PLoS Negl Trop Dis 16:e0010112. https://doi.org/10.1371/journal.pntd.0010112 Khan S, Sallard E (2023) Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms. Mol Biotechnol 65:196–205. https://doi.org/10.1007/s12033-022-00538-5 Kim HK, Song M, Lee J, Menon AV, Jung S, Kang YM, Choi JW, Woo E, Koh HC, Nam JW, Kim H (2017) In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nat Methods 14:153–159. https://doi.org/10.1038/nmeth.4104 Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869–874. https://doi.org/10.1038/nbt.3620 Lee Yu H, Cao Y, Lu X, Hsing I-M (2021) Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity. Biosens Bioelectron 189:113382. https://doi.org/10.1016/j.bios.2021.113382 Martin L, Rostami S, Rajan R (2023) Optimized protocols for the characterization of Cas12a activities, 1st edn. Elsevier Inc. Mohanraju P, Oost J, Jinek M, Swarts D (2018) Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein. Bio-Protocol 8:1–23. https://doi.org/10.21769/bioprotoc.2842 Morales-Moreno MD, Valdés-Galindo EG, Reza MM, Fiordelisio T, Peon J, Hernandez-Garcia A (2023) Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a. Cris J 6:116–126. https://doi.org/10.1089/crispr.2022.0074 Paenkaew S, Jaito N, Pradit W, Chomdej S, Nganvongpanit K, Siengdee P, Buddhachat K (2023) RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand. Vet Res Commun 47:1601–1613. https://doi.org/10.1007/s11259-023-10114-0 Paul B, Montoya G (2020) CRISPR-Cas12a: Functional overview and applications. Biomed J 43:8–17. https://doi.org/10.1016/j.bj.2019.10.005 Pouresmaeil M, Azizi-Dargahlou S (2023) Factors involved in heterologous expression of proteins in E. coli host. Arch Microbiol 205:212. https://doi.org/10.1007/s00203-023-03541-9 Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli : advances and challenges. Front Microbiol 5. https://doi.org/10.3389/fmicb.2014.00172 Shi Y, Fu X, Yin Y, Peng F, Yin X, Ke G, Zhang X (2021) CRISPR‐Cas12a System for Biosensing and Gene Regulation. Chem – An Asian J 16:857–867. https://doi.org/10.1002/asia.202100043 Strohkendl I, Saifuddin FA, Rybarski JR, Finkelstein IJ, Russell R (2018a) Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol Cell 71:816-824.e3. https://doi.org/10.1016/j.molcel.2018.06.043 Strohkendl I, Saifuddin FA, Rybarski JR, Finkelstein IJ, Russell R (2018b) Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol Cell 71:816-824.e3. https://doi.org/10.1016/j.molcel.2018.06.043 Swarts DC, Jinek M (2018) Cas9 versus Cas12a/Cpf1: Structure–function comparisons and implications for genome editing. WIREs RNA 9. https://doi.org/10.1002/wrna.1481 Wong MS, Wu S, Causey TB, Bennett GN, San K-Y (2008) Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production. Metab Eng 10:97–108. https://doi.org/10.1016/j.ymben.2007.10.003 Yang H, Wang H, Wang F, Zhang K, Qu J, Guan J, Shen W, Cao Y, Xia Y, Chen X (2022) Efficient extracellular production of recombinant proteins in E. coli via enhancing expression of dacA on the genome. J Ind Microbiol Biotechnol 49. https://doi.org/10.1093/jimb/kuac016 Yang Y, Wang D, Lü P, Ma S, Chen K (2023) Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system. Mol Biol Rep 50:3723–3738. https://doi.org/10.1007/s11033-023-08240-8 Zarzhitsky S, Jiang A, E. Stanley E, H. Hecht M (2020) Harnessing synthetic biology to enhance heterologous protein expression. Protein Sci 29:1698–1706. https://doi.org/10.1002/pro.3907 Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, Van Der Oost J, Regev A, Koonin E V., Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038 Zhang Z-X, Nong F-T, Wang Y-Z, Yan C-X, Gu Y, Song P, Sun X-M (2022) Strategies for efficient production of recombinant proteins in Escherichia coli : alleviating the host burden and enhancing protein activity. Microb Cell Fact 21:191. https://doi.org/10.1186/s12934-022-01917-y Supplementary Files SupplementaryMaterial.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor Revision 10 Jul, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers invited by journal 24 Jun, 2024 Editor assigned by journal 15 Jun, 2024 First submitted to journal 05 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4535821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318155224,"identity":"6e7e9465-7655-4f27-9ede-9ff070c14cc9","order_by":0,"name":"Orlando S. Goméz-Quintero","email":"","orcid":"","institution":"National Autonomous University of Mexico: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Orlando","middleName":"S.","lastName":"Goméz-Quintero","suffix":""},{"id":318155225,"identity":"5ffba4e3-0585-4f7e-bfe7-aa0c913a4326","order_by":1,"name":"Melissa D. Morales-Moreno","email":"","orcid":"","institution":"UNAM: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"D.","lastName":"Morales-Moreno","suffix":""},{"id":318155226,"identity":"9f2fd0a9-c397-473c-a15e-ced0ddb3357a","order_by":2,"name":"Erick G. Valdés-Galindo","email":"","orcid":"","institution":"UNAM: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Erick","middleName":"G.","lastName":"Valdés-Galindo","suffix":""},{"id":318155227,"identity":"dae61b1f-aefc-41a0-99ba-3b1a59869714","order_by":3,"name":"Rosa Elena Cárdenas-Guerra","email":"","orcid":"","institution":"UNAM: Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"Elena","lastName":"Cárdenas-Guerra","suffix":""},{"id":318155228,"identity":"4bdea4d2-97c1-4915-aea3-dae3f59f511e","order_by":4,"name":"Armando Hernández-García","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYNACNoYEBvbGBgYGAwYeErTwHCRZi0QCkYr5Z59O/FxQZpMnH/m48TZPwTYZ/vYDbNIFNdsYdGdgN0TiXO5m6Rnn0ooNbyc2W/MY3OaROJPAJj3j2G0GszMHsFtzhneDNG/b4cSNsxPbpEFaDBiAWnjYgFqON2DVIX+Gd/NvsJaZB6Fa+B8AtfwDajmM3RKDM7zbwLbMl2CEapEA2sLbhtsWQ6AWa55zaYkbeBKbLeeA/HLjYbM1b99tHlx+kQM67DZPmU3i/PbjD2+8+XPbnr8/+eBtnm+35cxuYA8xhAuBRkpAmIxgBxGOU/kGuJZRMApGwSgYBagAAEKHXa1pIB4KAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-2401-8139","institution":"National Autonomous University of Mexico Institute of Chemistry: Universidad Nacional Autonoma de Mexico Instituto de Quimica","correspondingAuthor":true,"prefix":"","firstName":"Armando","middleName":"","lastName":"Hernández-García","suffix":""}],"badges":[],"createdAt":"2024-06-05 18:29:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4535821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4535821/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60296608,"identity":"9c8bf4af-9b34-4c15-9b8c-d1773a6d6fea","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37977,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental details of the study. \u003cstrong\u003ea\u003c/strong\u003e General scheme of parameters evaluated to optimize the heterologous biosynthesis of AsCas12a protein in \u003cem\u003eE. coli\u003c/em\u003e \u003cstrong\u003eb\u003c/strong\u003e Plasmid pMBP-AsCas12a used for the production \u003cstrong\u003ec\u003c/strong\u003e Flow diagram of the experimental procedures implemented for protein expression\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/414748ba28fba4222b4ad1ee.png"},{"id":60296609,"identity":"1db65071-4037-444a-8d24-873dedeb4948","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162794,"visible":true,"origin":"","legend":"\u003cp\u003eIPTG concentration effect on AsCas12a expression in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). \u003cstrong\u003ea\u003c/strong\u003e SDS-PAGE of lysed bacteria incubated at 12°C and 24h after induction with IPTG. \u003cstrong\u003eb\u003c/strong\u003e Densitometric analysis of percentage protein from SDS-PAGE in \u003cstrong\u003ea\u003c/strong\u003e Arrow points towards AsCas12a protein. **** P =\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/11a6ec0f31f2f62245d4703b.png"},{"id":60296603,"identity":"f8259858-34fc-4b46-aa90-40fb84cac5ba","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":127398,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature effect on protein AsCas12a expression in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (9h induction with 0.3 mM IPTG). \u003cstrong\u003ea\u003c/strong\u003e SDS-PAGE of lysed bacteria. \u003cstrong\u003eb\u003c/strong\u003e Densitometric analysis of percentage protein from SDS-PAGE in \u003cstrong\u003ea\u003c/strong\u003e Arrows point towards AsCas12a protein. **** P =\u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/0df39b031ba83e4cb83bc01f.png"},{"id":60297413,"identity":"a6c05993-6ab4-4960-9775-94e3a6469202","added_by":"auto","created_at":"2024-07-15 09:59:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141534,"visible":true,"origin":"","legend":"\u003cp\u003eCulture media and 1% glucose impact on AsCas12a expression in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (9h of induction with 0.3 mM IPTG at 30°C). \u003cstrong\u003ea\u003c/strong\u003e SDS-PAGE of lysed bacteria cultured in different media. \u003cstrong\u003eb\u003c/strong\u003e Densitometric analysis of percentage protein from SDS-PAGE in \u003cstrong\u003ea.\u003c/strong\u003e Arrows point towards AsCas12a protein. ** P = 0.005\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/4cfc758fed6bc00b60e5a697.png"},{"id":60296604,"identity":"989b384d-d87e-403c-906a-0cda69562781","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e strain impact on recombinant AsCas12a production. \u003cstrong\u003ea\u003c/strong\u003eSDS-PAGE of lysed bacteria grown in TB + 1% glucose for 9h at 30°C and induced with 0.3 mM IPTG. \u003cstrong\u003eb\u003c/strong\u003e Densitometric analysis of percentage protein from SDS-PAGE in (a). Arrows point towards AsCas12a protein. **** P \u0026lt; 0.0001 and *** P = 0.0005\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/6c914108f696d1cf83f9ed55.png"},{"id":60296606,"identity":"e643b8d5-b8ed-4f80-9d8d-9f79ee984a51","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":148035,"visible":true,"origin":"","legend":"\u003cp\u003eHeterologous production of AsCas12a protein under optimized and non-optimized conditions. \u003cstrong\u003ea\u003c/strong\u003e Bacterial optical density kinetics. \u003cstrong\u003eb\u003c/strong\u003eTotal soluble protein production kinetics determined by BCA. \u003cstrong\u003ec\u003c/strong\u003e SDS-PAGE of lysed bacteria at specific times. kinetics of percentage \u003cstrong\u003ed\u003c/strong\u003e and total \u003cstrong\u003ee\u003c/strong\u003eAsCas12a protein production in soluble fraction calculated from SDS-PAGE in \u003cstrong\u003ec.\u003c/strong\u003eArrow points towards AsCas12a protein. Dashed lines are to guide the eye\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/d6f9de694b1ff2d91f805b5d.png"},{"id":60297414,"identity":"62520d0b-6d10-42f8-baa9-3727e4905045","added_by":"auto","created_at":"2024-07-15 09:59:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":111108,"visible":true,"origin":"","legend":"\u003cp\u003eAsCas12a purification and functional evaluation. \u003cstrong\u003ea\u003c/strong\u003e Purification process of AsCas12a protein. \u003cstrong\u003eb\u003c/strong\u003e \u003cem\u003eCis\u003c/em\u003e-cleavage assay in a 1% agarose gel using target pET28a-SARS-CoV-2 pDNA(+) and a non-target pDNA(-). \u003cstrong\u003ec\u003c/strong\u003e \u003cem\u003eTrans\u003c/em\u003e-cleavage assay using FAM-ssDNA-quencher as fluorescence reporter. RNP: AsCas12a + gRNA.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/164c404a41285170e23dc4e8.png"},{"id":60297877,"identity":"56aaec63-c8c9-4950-9af3-5c852ae889f5","added_by":"auto","created_at":"2024-07-15 10:07:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1594006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/99689243-4aa7-4a72-ba43-7e62aaf6f8b3.pdf"},{"id":60296611,"identity":"6113ce90-815a-4513-aae5-0a31b3b390cf","added_by":"auto","created_at":"2024-07-15 09:51:54","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3567662,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535821/v1/2efa9b596b90a4c2536b1719.pdf"}],"financialInterests":"","formattedTitle":"Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli","fulltext":[{"header":"Key Points","content":"\u003col\u003e\n \u003cli\u003eLab-scale production of Cas12a was highly boosted using a simple strategy.\u003c/li\u003e\n \u003cli\u003eOptimized conditions increased AsCas12a production yield up to 4.5 times.\u003c/li\u003e\n \u003cli\u003eMethod offers efficient, straightforward and high-yield Cas12a production.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe CRISPR-Cas12a system, formerly known as Cpf1, stands as a potent molecular tool with diverse applications in genome editing, molecular diagnostics, and various biotechnological and biomedical fields (Paul and Montoya \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khan and Sallard \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Given the manifold applications of the CRISPR-Cas12a system, there exists a widespread interest in the small-scale, high-yield biosynthesis of the Cas12a protein among research laboratories in both developed and non-developed countries (Bandyopadhyay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jirawannaporn et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Paenkaew et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Many laboratories around the world need a continuous supply of the protein to carry on and continue with their research activities. Although Cas12a protein is available commercially, its high cost makes research labs to prefer to produce and purify themselves the protein. However, the current available lab protocols are low efficiency and have not been optimized. Despite the increasing application of Cas12a, there remains a noticeable lack of studies focused on its small lab-scale production without the need to use a bioreactor or specialized bacterial strains. While some groups have reported the recombinant production of \u003cem\u003eFrancisella novicida\u003c/em\u003e Cas12a protein (Zetsche et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Strohkendl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), only very few reports (Mohanraju et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) have described detailed protocols for the biosynthesis of other relevant orthologues such as \u003cem\u003eAcidaminococcus sp\u003c/em\u003e Cas12a protein (AsCas12a). Surprisingly, there is a notable gap in the literature regarding the systematic study of production of Cas12a, underscoring the need for comprehensive investigations in this area. Consequently, an exploration of the recombinant production of Cas12a proteins is imperative to advance CRISPR-Cas12a-based research, with a particular focus on the AsCas12a variant in this study.\u003c/p\u003e \u003cp\u003eThe endonuclease AsCas12a is classified as a type V CRISPR-Cas system, as it serves as the sole effector protein responsible for DNA cleavage. Cas12a possesses key attributes that position it as a robust alternative to the widely utilized CRISPR-Cas9 system (Swarts and Jinek \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, the crRNA of Cas12a is shorter than Cas9 gRNA (40\u0026ndash;44 vs 100 nt), and it cleaves dsDNA in a staggered manner. Cas12a exhibits a higher precision in binding to dsDNA sequences compared to Cas9, boasting a substantial affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e ~fM) and displays a pronounced \u003cem\u003etrans\u003c/em\u003e-cleavage activity upon ssDNA (commonly referred to as collateral activity) (Jinek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zetsche et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These distinctive features make the Cas12a protein an excellent choice within the CRISPR-Cas systems for both fundamental and applied research.\u003c/p\u003e \u003cp\u003eAsCas12a forms a ribonucleoprotein complex (RNP) in conjunction with a CRISPR RNA (crRNA, also known as guide RNA or gRNA). The RNP makes a highly specific cleavage of both DNA strands upon binding the target DNA with a remarkable affinity (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e of 54 fM) (Zetsche et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Strohkendl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Particularly effective with T-rich genomes, AsCas12a recognizes the PAM 5\u0026rsquo; TTTV 3\u0026rsquo; (Kim et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Comprising 1,307 amino acid residues and a molecular weight of 151.2 kDa, the AsCas12a is structured into REC and NUC lobes (Dong et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The NUC lobe contains the RuvC-I, II and III domains, responsible for the ultimate cleavage of dsDNA. Distinct from SpyCas9, AsCas12a exhibits unique features (Swarts and Jinek \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). AsCas12a uses a shorter guide RNA, lacks a tracrRNA, cleaves dsDNA with 5\u0026rsquo; overhangs and has a longer distance between the seed sequence and the cleavage site (Swarts and Jinek \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite these differences, AsCas12a robustly and efficiently cleaves dsDNA in human cells (Zetsche et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), displaying on-target efficiencies comparable to the widely used SpyCas9 (Kleinstiver et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All this underscores the need of studies focused on optimizing the recombinant production of AsCas12a in \u003cem\u003eE. coli\u003c/em\u003e, the most commonly employed heterologous expression system of choice for many research labs around the world (Rosano and Ceccarelli \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study presents a systematic study aimed to identify optimal conditions for the lab-scale production of recombinant AsCas12a in \u003cem\u003eE. coli\u003c/em\u003e as expression system, comparing them with initial conditions based on standard available protocols (Mohanraju et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was studied the effect the influence of several parameters, including \u003cem\u003eE. coli\u003c/em\u003e strain, IPTG concentration, culture media, media supplementation with glucose, and post-induction temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The protein expression was monitored under various conditions, and the most effective combinations were identified to ensure efficient protein expression. The significance of studies that report improved conditions to express and purify CRISPR-Cas proteins is of large relevance, as they contribute to advancing the entire CRISPR-Cas field by facilitating the availability of proteins for experimentation. It was considered that the optimized conditions for the heterologous production of AsCas12a protein in \u003cem\u003eE. coli\u003c/em\u003e will prove beneficial to research laboratories engaged in CRISPR-based studies that have available basic equipment and materials for heterologous bacterial expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003eThe pMBP-AsCas12a plasmid was employed for the production of AsCas12a protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), generously provided by Prof. Ilya Finkelstein at the University of Texas-Austin (Addgene #113430) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This plasmid harbors an ampicillin resistance gene and the AsCas12a gene, featuring a His10-tag, all under the control of a T7 promoter. The plasmid pPET28a-SARS-CoV-2-geneN was utilized for \u003cem\u003ecis\u003c/em\u003e and \u003cem\u003etrans\u003c/em\u003e cleavage assays of the AsCas12a protein, a generous gift from Dr. Luis Brieba de Castro from LANGEBIO-UGA, Mexico, containing the N gene from the SARS-CoV-2 Wuhan reference genome.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe Luria Bertani (LB) Broth culture medium was purchased from Invitrogen (Waltham, Massachusetts, USA). The highly enriched Terrific Broth (TB) medium was prepared with 24 g/L yeast extract (Formedium, Hunstanton, UK), 12 g/L Tryptone (Sigma-Aldrich, St. Louis, Missouri, USA), 4 mL glycerol (JT Baker, Phillipsburg, NJ, USA), and 0.17 M phosphate buffer (pH 7) (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, Sigma-Aldrich). Glucose, ampicillin, kanamycin, 2-Mercaptoethanol and bromophenol blue were purchased from Sigma-Aldrich. Isopropil \u0026szlig;-D-1-thiogalactopyranoside (IPTG) was obtained from Invitrogen (Carlsbad, USA). Agar, acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate, and glycine were purchased from Biorad (Hercules, California, United States). N, N, N\u0026prime;, N\u0026prime;-Tetramethyl ethylenediamine (TEMED) was acquired from IBI Scientific (Dubuque, Iowa, United States), while Tris was obtained from Gold Bio (St. Louis, Missouri, United States). Furthermore, gRNAs and ssDNA reporter probe were synthesized by Integrated DNA Technologies-IDT (Coralville, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and culture media preparation\u003c/h2\u003e \u003cp\u003eThe strains employed in the assays for recombinant protein biosynthesis, BL21 (DE3), Rosetta (DE3), BL21(pLysS), Tuner\u003csup\u003e\u0026trade;\u003c/sup\u003e (DE3), and NiCo (DE3), were generously provided by Dr. Patricia Cano S\u0026aacute;nchez and Dr. Corina-Diana Ceapă from the Institute of Chemistry, UNAM, Mexico. The LB broth was prepared by dissolving 12.5 g of LB media powder in 1 L of distilled water. TB solution was prepared as mentioned above. These media solutions were sterilized using an autoclave (Sterilite 24, Fisher Scientific, Waltham, USA). As needed, LB and TB media were enriched by supplementing with glucose, reaching a final concentration of 1% (w/v), previously sterilized using 0.2 \u0026micro;m filters (Sartorius, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBacterial transformation and plasmid production\u003c/h2\u003e \u003cp\u003eBacterial transformation was achieved through the thermal shock method. Competent \u003cem\u003eE. coli\u003c/em\u003e DH5α (or any other strain) were placed on ice for 5 min with 2 \u0026micro;L plasmid. The mixture underwent a temperature shift to 42\u0026deg;C for 30 s, followed by an incubation on ice for 5 min. The sample then was mixed with 100 \u0026micro;L LB and incubated at 37\u0026deg;C for 1 h. The mixture was added to agar plates supplemented with 100 \u0026micro;g/mL ampicillin and incubated overnight at 37\u0026deg;C. A colony of bacteria transformed with the plasmid was grown overnight in 5 mL LB medium, incorporating the necessary antibiotic (ampicillin or kanamycin) at 100 \u0026micro;g/mL. Subsequently, plasmids were purified from the culture using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExpression of protein AsCas12a\u003c/h2\u003e \u003cp\u003eThe initial experimental conditions were based on previously published protocols for the expression of Cas12a (Mohanraju et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Strohkendl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To initiate the pre-inoculum, a colony was selected from an agar plate and cultivated in a 250 mL Erlenmeyer flask containing 100 mL media (LB or TB, depending on the assay) supplemented with 100 \u0026micro;g/mL of ampicillin. The pre-inoculum was incubated at 37\u0026deg;C for 16 h and 225 rpm using a MaxQ 4,000 shaker incubator equipped with controlled temperature settings (Thermo Scientific, Waltham, Massachusetts). Following this, the pre-inoculum was diluted to an optical density (OD) of 0.1 in 100 mL of culture media (TB or LB, with or without 1% glucose, incorporating 100 \u0026micro;g/mL of ampicillin). The culture was then incubated at 37\u0026deg;C with 225 rpm until the OD reached a value of 0.6\u0026ndash;0.8 (approximately 1.5 h). OD was measured in a BioPhotometer plus spectrophotometer (Eppendorf, Hamburg, Germany). Then, the culture was incubated at the desired experimental temperature (12, 18, 30 or 37\u0026deg;C) with 225 rpm. After 30 min, IPTG induction was carried out (0.3, 0.6, 1, 1.3, 1.6 mM), and the culture was incubated for the designated time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell lysis and sample preparation\u003c/h2\u003e \u003cp\u003eBacterial samples taken during the growth curves were treated in the following way before their analysis of protein expression. 1 mL of bacterial culture was centrifuged at 4\u0026deg;C for 5 min at 13,000 rpm using a microcentrifuge AccuSpin Micro 17R (Fisher Scientific, Waltham, USA). The supernatant was discarded, and the pellet was resuspended in 100 \u0026micro;L cell lysis buffer [200 mM 2-Mercaptoethanol, 25% glycerol, 10% SDS, 0.5 M Tris-HCl (pH 6.8), and 0.5% bromophenol blue] and heated up to 95\u0026deg;C for 5 min using an Isotemp Thermoblock (Fisher Scientific, Waltham, USA). Finally, the solution was centrifuged at 4\u0026deg;C for 5 min at 13,000 rpm and the supernatant was recovered and stored at -20\u0026deg;C for further analysis, then only the soluble fraction was further analyzed. Cell lysis buffer without bromophenol blue was used when total protein concentration present in the bacteria culture was measured by Nanodrop. When very dense cultures were lysed, they were centrifugated during 55 min to eliminate the excess of debris.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of total soluble protein\u003c/h2\u003e \u003cp\u003eThe total protein quantification was performed using a NanoDrop One UV-Vis spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and by micro-bicinchoninic acid assay (BCA) using the Pierce\u0026trade; BCA Protein Assay Kit (ThermoScientific). For NanoDrop, approximately 1.5 \u0026micro;L of the cell lysate were scanned using the protein A280 program after a buffer baseline correction determined by the device. For micro\u0026ndash;BCA microplate Assay 150 \u0026micro;L of sample (2 \u0026micro;L of bacterial lysate diluted to a volume of 500 \u0026micro;L) were loaded into a 96-well microplate by triplicate together with the albumin standard (prepared following the instructions of the protocol). Then, 150 \u0026micro;L of the BCA working reagent was added to each well and the mixture was incubated for 2h at 37\u0026deg;C to finally read in a Cytation 5 Multi Mode Reader (BioTek, Santa Clara, USA) at 562 nm. The obtained results were analyzed and plotted using GraphPad 10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE analysis\u003c/h2\u003e \u003cp\u003eProtein expression from lysed samples was analyzed by electrophoresis in SDS-PAGE gel using a 247.6 mM Tris-glycine (pH 8) as running buffer at 160 V for 45 min. Precision Plus Protein\u0026trade; Kaleidoscope\u0026trade; Prestained Protein Standard was used as a molecular weight marker (Bio Rad, Hercules, California, United States). Gels were water-washed three times and then stained with SimplyBlue\u0026trade; Safe Stain (Thermo Fisher, Waltham, USA). Gels were imaged in an Azure 200 Gel Imager (Azure Biosystems, California, USA). Densitometric analysis was carried out using ImageJ software. The percentage of AsCas12a protein present in each sample was determined by measuring the intensity of the band corresponding to the AsCas12a protein band relative to the total protein band intensity present in the lane (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChromatography protein purification\u003c/h2\u003e \u003cp\u003eProtein purification was performed following a previously reported purification protocol (Morales-Moreno et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The lysed product from 1 L was purified in an ӒKTA pure\u0026trade; chromatography system using affinity, ionic exchange, and molecular size exclusion chromatography. Metal affinity chromatography was done with a HisTrapTM HP 5 mL column, and the protein was eluted with 250 mM imidazole at a flow rate of 2 mL/min The Histidine tag was cleaved off using TEV protease during an overnight dialysis at 4\u0026deg;C. The ionic exchange chromatography was carried out using a HiTrap Heparin HP 5 mL column and the protein was eluted with IEx-B buffer (2M potassium chloride) at a flow rate of 2 mL/min. Finally, the molecular size exclusion chromatography was performed using a HiLoad 16/600 Superdex 200 pg 125 mL column and at a buffer flow rate of 0.5 mL/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCis-cleavage activity on pDNA\u003c/h2\u003e \u003cp\u003eAsCas12a:crRNA ribonucleoprotein complexes (RNPs) were assembled by incubating for 30 min at 37\u0026deg;C a 1:1 molar mixture of the purified AsCas12a protein and crRNA designed towards the N gene (Morales-Moreno et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) of SARS-CoV-2 in 1X NEB 2.1 buffer (New England Biolabs, Ipswich, Massachusetts, USA). Then, RNP complexes were mixed with the target dsDNA (pPET28a-SARS-CoV-2-geneN) to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively (molar proportion RNP:pDNA of 10:1) and incubated in a thermoblock at 37\u0026deg;C during 1 h. Then, samples were mixed with Gel Loading Dye 6X (Invitrogen) and evaluated in an 1% agarose gel (Thermo Fisher) using TAE 1X as running buffer (Thermo Fisher) at 140 V for 45 min after staining with SYBR\u0026trade; Safe DNA Gel Stain (Invitrogen). Gels were imaged with an Azure 300 gel imager system (Azure Biosystems, Dublin, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTrans-cleavage activity on ssDNA\u003c/h2\u003e \u003cp\u003e \u003cem\u003eTrans\u003c/em\u003e-cleavage activity assay was evaluated following a previous reported protocol (Morales-Moreno et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In short, RNPs were assembled as previously indicated and mixed with the target pPET28a-SARS-CoV-2-geneN to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively in 1X NEB 2.1 buffer. As a reporter probe 250 nM of ssDNA attached to FAM and a quencher was used. \u003cem\u003eTrans\u003c/em\u003e-cleavage activity on ssDNA was followed by fluorescence at 37\u0026deg;C during 1 h using a Cytation 5 Cell Imaging Multi Mode Reader (BioTek, Santa Clara, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical evaluations\u003c/h2\u003e \u003cp\u003eOrdinary one-way ANOVA tests were conducted using the GraphPad Prism 10 software on all the SDS-PAGE densitometry assays results of the supernatants obtained from the lysed bacteria of the cultures (IPTG concentration, Temperature, Culture Media, Glucose supplementation and \u003cem\u003eE. coli\u003c/em\u003e Strain) to compare if there was a significant difference between the original parameters and the new experimental parameters. Values of P greater than 0.5 were considered as non-significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of IPTG concentration\u003c/h2\u003e \u003cp\u003eFirst, it was explored a 0.3 to 1.6 mM of IPTG concentrations over a 24 h induction period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The level of AsCas12a protein production upon IPTG concentration was followed by analyzing cell lysates in SDS-PAGE. The SDS-PAGE illustrated successful induction of AsCas12a at all tested IPTG levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Fig. S2). Densitometric analysis revealed a decreasing trend in the total AsCas12a protein band percentage relative to the total band intensity with increasing IPTG concentrations (21.1, 19.9, 18.9, 20.3, and 17.1% for 0.3, 0.6, 1, 1.3, and 1.6 mM IPTG, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, the differences were not statistically significant between them.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of temperature on AsCas12a expression\u003c/h2\u003e \u003cp\u003eTo assess the impact of temperature on protein expression, recombinant \u003cem\u003eE. coli\u003c/em\u003e cultures induced with 0.3 mM IPTG were incubated at various temperatures. After 9 h of induction, cell lysates were analyzed by SDS-PAGE to observe the percentage of produced AsCas12a protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplementary Fig. S3). AsCas12a protein expression was observed across all temperatures, albeit in varying amounts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Densitometric analysis revealed that the highest production of AsCas12a proteins was approximately 27% (of the total soluble protein) at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This contrasts with 12.2% (12\u0026deg;C), 17.9% (18\u0026deg;C), and 13.7% (37\u0026deg;C). The percentage of protein at 30\u0026deg;C represents a\u0026thinsp;~\u0026thinsp;2.2-fold and ~\u0026thinsp;1.5-fold increase compared to 12\u0026deg;C and 18\u0026deg;C, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of culture media\u003c/h2\u003e \u003cp\u003eWe investigated the impact of highly accessible media, such as LB and TB, for AsCas12a production, and assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After inducing AsCas12a expression with 0.3 mM IPTG and incubating 30\u0026deg;C for 9 h, the percentage of AsCas12a in the total soluble protein (present in cell lysate) was measured with SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. S4). After comparing LB and TB, it was observed a\u0026thinsp;~\u0026thinsp;1.6-fold improvement (16.9 and 27.2%, respectively). However, supplementing TB with 1% glucose increased AsCas12a protein production from 27.2 to 31.5%, whereas LB\u0026thinsp;+\u0026thinsp;1% glucose reduced from 16.9 to 15.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Changing LB to TB\u0026thinsp;+\u0026thinsp;1% glucose represented a\u0026thinsp;~\u0026thinsp;1.9-fold increment. Notably, TB\u0026thinsp;+\u0026thinsp;1% glucose production was high enough that the lysed sample caused streaks on SDS-PAGE bands.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of strain\u003c/h2\u003e \u003cp\u003eEmploying five commonly used \u003cem\u003eE. coli\u003c/em\u003e DE3 strains (BL21, Tuner, Rosetta, PlysS, and NiCo), we aimed to identify the most efficient one for AsCas12a biosynthesis under our previously optimized conditions (TB\u0026thinsp;+\u0026thinsp;1% glucose, 0.3 mM IPTG, and 30\u0026deg;C). After analyzing cell lysates with SDS-PAGE, it was revealed distinct protein production levels among the strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Fig. S5). BL21 exhibited prominent production, followed by Rosetta and NiCo, while pLysS and Tuner showed the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Anomalous electrophoretic mobility was observed in bands with high protein expression, notably in BL21 and Rosetta. The AsCas12a protein production was quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The quantitative analysis confirmed BL21 as the top performer, 31.5% from the total soluble protein corresponded to AsCas12a protein, followed by Rosetta with 26.7%, NiCo 18.2%, Tuner 15.5%, and pLysS with 14.0%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eComparison between initial and improved conditions\u003c/h2\u003e \u003cp\u003eNext, it was assessed with detail the impact of optimized conditions (BL21(DE3) strain, TB\u0026thinsp;+\u0026thinsp;1% glucose, 0.3 mM IPTG, and 30\u0026deg;C) and compared against initial non-optimized conditions (BL21(DE3) strain, LB, 1 mM IPTG, and 12\u0026deg;C). This was done by following over 24 h (post-IPTG induction) cell growth kinetics, production of total soluble protein, and production of AsCas12a protein in both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter 9h, optimized conditions exhibited significantly faster bacterial growth, reaching a maximum OD of 6.6, whereas non-optimized conditions exhibited an OD of 2.1 absorbance units. It was only after 23h that the non-optimized conditions reached an OD of 2.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). A similar trend was observed for amount of total soluble protein (determined by BCA). After ~\u0026thinsp;9h the optimized conditions reached\u0026thinsp;~\u0026thinsp;15 mg/mL (relative to volume of bacterial lysate), whereas for non-optimized conditions was ~\u0026thinsp;10 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Even after 24 h, protein production in non-optimized conditions was still lower (~\u0026thinsp;11.5 mg/mL).\u003c/p\u003e \u003cp\u003eTo correlate the previous results of production of total soluble protein with AsCas12a protein production, SDS-PAGE and densitometric analysis were carried out (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E, Supplementary Fig. S6). The quantitative analysis of SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) confirmed that ~\u0026thinsp;25% of the total soluble protein produced at 9 h corresponded to AsCas12a protein, remaining constant thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). In contrast, non-optimized conditions showed a slower increase, reaching\u0026thinsp;~\u0026thinsp;13% of AsCas12a protein at 9h and ~\u0026thinsp;16% only until 24 h. All this means that the concentration of AsCas12a protein produced at 9 h for optimized conditions was ~\u0026thinsp;3.5 mg/mL (relative to volume of cell lysate), and ~\u0026thinsp;1.2 mg/mL for non-optimized conditions, representing a\u0026thinsp;~\u0026thinsp;3-fold increment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). A summary of all values is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the results obtained between optimized and non-optimized conditions in bacterial lysates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eOptical Density\u003c/p\u003e \u003cp\u003e(A.U.)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eTotal Soluble Protein\u003c/p\u003e \u003cp\u003e(mg/mL)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eAsCas12a\u003c/p\u003e \u003cp\u003e(%)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eAsCas12a\u003c/p\u003e \u003cp\u003e(mg/mL)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNon-optimized conditions\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOptimized conditions\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e24.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eBacterial growth measured as optical density.\u003c/p\u003e \u003cp\u003e \u003csup\u003eb\u003c/sup\u003eTotal amount of cleared soluble protein per volume of bacterial lysate determined by BCA.\u003c/p\u003e \u003cp\u003e \u003csup\u003ec\u003c/sup\u003ePercentage of protein AsCas12a in total soluble protein determined by densitometry in SDS-PAGE.\u003c/p\u003e \u003cp\u003e \u003csup\u003ed\u003c/sup\u003eTotal amount of protein AsCas12a produced in the bacterial lysate determined from SDS-PAGE and BCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePurification and functional evaluation of AsCas12a protein\u003c/h2\u003e \u003cp\u003eTo assess the functionality of the recombinant AsCas12a produced under optimized conditions, first the protein AsCas12a was purified and then we examined its \u003cem\u003ecis\u003c/em\u003e- and \u003cem\u003etrans\u003c/em\u003e-cleavage activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe protein was expressed under optimized and non-optimized conditions in 1 L flasks, purified with Ni-NTA affinity, ionic exchange, and molecular size exclusion chromatography (Morales-Moreno et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and characterized by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). After purification the production yield of AsCas12a protein was 23.4 mg/L (of bacterial culture), whereas for non-optimized conditions was 5.2 mg/L. Western blot confirmed its identity and successful purification (Supplementary Fig. S7).\u003c/p\u003e \u003cp\u003eThen, it was tested the functional performance of the AsCas12a protein to cleave dsDNA, \u003cem\u003ecis\u003c/em\u003e-activity, (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The \u003cem\u003ecis\u003c/em\u003e-cleavage activity of RNP complex (AsCas12a: crRNA) efficiently digested the supercoiled target pDNA (7,253 kbp), while leaving intact the non-target pDNA (9,210 kbp). Finally, it was evaluated the \u003cem\u003etrans\u003c/em\u003e-cleavage activity on ssDNA, also called collateral activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). It was used two gRNAs that recognize the sequence of the nucleocapsid gene of SARS-CoV-2 located in a pDNA. To evaluate the functional performance of AsCas12a protein, it was followed the fluorescence increase upon cleavage of a FAM-quencher labeled reporter ssDNA after recognition of the target pDNA sequence. The correct \u003cem\u003etrans\u003c/em\u003e-cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRISPR-Cas systems have become one of the most relevant biomolecular systems for current biotechnological research in many labs around the world. Warrantying a continuous and cheap supply is enormously important for doing basic research and technology development in a productive and timely manner. The system CRISPR-Cas12a is the second most used just below CRISR-Cas9 (Paul and Montoya \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Besides genetic engineering, Cas12a has ample applications in biosensor development (Shi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hernandez-Garcia et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In particular, AsCas12a is one of the most used between the various homologous of Cas12a. AsCas12a protein is commonly produced in \u003cem\u003eEscherichia coli\u003c/em\u003e; however, an efficient production of the protein has not been reported. Having an optimized production of protein AsCas12a at lab scale would benefit many research labs that continuously use it. Furthermore, optimal production of protein AsCas12a has more chances of being adopted if is based on materials that are easily accessible and available to most research labs, also, if the optimization is based on simple parameters. That is why here we investigated the effect of multiple simple parameters that are important for the efficient production of AsCas12a protein.\u003c/p\u003e \u003cp\u003eFirst, we explored the effect of 0.3 to 1.6 mM of IPTG concentration over a 24 h induction period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Our results demonstrated that IPTG concentrations above 0.3 mM had not significant impact. This suggests that 0.3 mM concentration is enough to effectively induce AsCas12a expression in \u003cem\u003eE. coli\u003c/em\u003e. This IPTG concentration is very close to previously reported values of 0.13 and 0.2 mM IPTG (Jinek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mohanraju et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); but contrasts with the 1 mM used by others (Strohkendl et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNext, we investigated the effect of temperature on AsCas12a expression. After assessing the impact of various temperatures (12, 18, 30 and 37\u0026ordm;C) on protein expression in recombinant \u003cem\u003eE. coli\u003c/em\u003e cultures induced with 0.3 mM IPTG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we found that 30\u0026deg;C was the best post-induction temperature for successful enhancement of protein production. Producing at 30\u0026deg;C increased\u0026thinsp;~\u0026thinsp;2.2-fold and ~\u0026thinsp;1.5-fold the production of AsCas12a protein compared to 12\u0026deg;C and 18\u0026deg;C, respectively. This finding is significant since most of the protocols reported for producing AsCas12a using \u003cem\u003eE. coli\u003c/em\u003e commonly used temperatures of 12 or 18\u0026ordm;C but not 30\u0026ordm;C (Jinek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mohanraju et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Martin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther aspect of interest in our study was to evaluate the effect of culture media. Analyzing effect of culture media is crucial for optimizing recombinant protein expression (Rosano and Ceccarelli \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). For that reason, we investigated the impact of highly available media, such as LB and TB, for AsCas12a production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Also, we assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). TB supplemented with 1% glucose resulted advantageous for recombinant production of protein AsCas12a, even surpassing LB, LB\u0026thinsp;+\u0026thinsp;1% glucose, and TB alone in protein production. The impact on production of adding glucose to TB was very relevant. This positive effect may stem from the role of glucose as an alternative carbon source to an already rich carbon source media (TB contains glycerol), accelerating total biomass and protein production (Choi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Glycerol in TB also aids in preventing acetate formation and serves as an alternative carbon source. Conversely, the negative effect of glucose in LB may be attributed to its transformation into acetates, hindering growth, inhibiting protein formation, and diverting carbon away from biomass to protein product (Wong et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe final parameter was the bacterial strain. The choice of \u003cem\u003eE. coli\u003c/em\u003e strain may significantly influence the recombinant production of protein AsCas12a since it has been observed that \u003cem\u003eE. coli\u003c/em\u003e strains play important roles for heterologous protein production (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pouresmaeil and Azizi-Dargahlou \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among all the studied strains, BL21(DE3) resulted the most efficient (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This is positive judging that is a very common strain in many biotechnological research labs. On the other hand, it could be interesting to explore new \u003cem\u003eE. coli\u003c/em\u003e strains that are being developed for efficient heterologous production of proteins under the strategies of synthetic biology (Zarzhitsky et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, we compared between initial and improved conditions. We assessed in detail the impact of combining our best-found conditions against initial non-optimized conditions which were based of typical reported protocols for Cas12a protein production (Martin et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mohanraju et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Strohkendl et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). The higher values obtained for OD and concentration of total soluble protein and total AsCas12a protein can tell us that our optimization strategy was successful (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Of particular relevance is the production of AsCas12a. The concentration of AsCas12a protein produced at 9 h for optimized conditions was ~\u0026thinsp;3.5 mg/mL (relative to volume of cell lysate), which contrast with ~\u0026thinsp;1.2 mg/mL obtained with non-optimized conditions. The difference represents a\u0026thinsp;~\u0026thinsp;3-fold increment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). The striking differences between both conditions could be explained by the usage of the rich media TB\u0026thinsp;+\u0026thinsp;1% glucose and a higher incubation temperature for the optimized conditions. The efficiency of optimized conditions became evident, indicating their suitability for high-level AsCas12a protein production. In fact, the non-optimized conditions will require much longer than 24 h to produce similar levels of protein. All this demonstrates that our optimized conditions are producing more protein in shorter time.\u003c/p\u003e \u003cp\u003eVerifying the functional performance of the produced and purified AsCas12a protein is of outmost importance. We could verify the cleavage of dsDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), \u003cem\u003ecis\u003c/em\u003e-activity, which one of the earliest and most common application of AsCas12a (Zetsche et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kleinstiver et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bandyopadhyay et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Also, the evaluation of the \u003cem\u003etrans\u003c/em\u003e-cleavage activity on ssDNA, also called collateral activity, is crucial since it is the base of multiple methods for genetic detection and clinical diagnostics in labs around the world (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lee Yu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jirawannaporn et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Morales-Moreno et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Paenkaew et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The \u003cem\u003etrans\u003c/em\u003e-cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), showcasing the protein full collateral activity and ability to detect specific DNA sequences. Both activities demonstrated to work between acceptable standards.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identified several parameters that significantly enhanced the heterologous lab-scale production of AsCas12a protein in recombinant \u003cem\u003eE. coli\u003c/em\u003e cultures. Optimized conditions were found for all the studied parameters (IPTG concentration, induction temperature, culture media, and type of bacterial strain). Combination of all optimized conditions increased\u0026thinsp;~\u0026thinsp;3 times the bacterial production of protein AsCas12a in a shorter time (~\u0026thinsp;9h). The protein production yield after purification was 23.4 mg/L of culture volume, representing a 4.5-fold increase in comparison to the non-optimized conditions. Importantly, the purified AsCas12a protein retained full \u003cem\u003ecis\u003c/em\u003e and \u003cem\u003etrans\u003c/em\u003e cleavage activities, which are essential for various downstream CRISPR-Cas applications. These optimized protein production conditions offer a valuable resource for research labs seeking higher yields of functional AsCas12a protein, all in a more efficient timeframe and using a simple protocol that requires accessible materials and equipment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by UNAM-PAPIIT (IN210121 \u0026amp; IV200820).\u0026nbsp;The authors gratefully acknowledge support from Agencia Mexicana de Cooperacion Internacional para el Desarrollo (AMEXCID)\u0026mdash;Secretaria de Relaciones Exteriores Mexico (Proyectos COVID-19).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest for this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability (data transparency):\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSupplementary information is included.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOSQG and AHG conceived and designed research. OSQG, MDMM, EGVG and RECG conducted experiments. OSQG and AHG analyzed data. AHG wrote the manuscript with input from OSQG. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBandyopadhyay A, Kancharla N, Javalkote VS, Dasgupta S, Brutnell TP (2020) CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement. Front Plant Sci 11. https://doi.org/10.3389/fpls.2020.584151\u003c/li\u003e\n\u003cli\u003eCarmignotto GP, Azzoni AR (2019) On the expression of recombinant Cas9 protein in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) and BL21(DE3) Rosetta strains. J Biotechnol 306:62\u0026ndash;70. https://doi.org/10.1016/j.jbiotec.2019.09.012\u003c/li\u003e\n\u003cli\u003eChen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (80- ) 360:436\u0026ndash;439. https://doi.org/10.1126/science.aar6245\u003c/li\u003e\n\u003cli\u003eChoi JH, Keum KC, Lee SY (2006) Production of recombinant proteins by high cell density culture of \u003cem\u003eEscherichia coli\u003c/em\u003e. Chem Eng Sci 61:876\u0026ndash;885. https://doi.org/10.1016/j.ces.2005.03.031\u003c/li\u003e\n\u003cli\u003eDong D, Ren K, Qiu X, Zheng J, Guo M, Guan X, Liu H, Li N, Zhang B, Yang D, Ma C, Wang S, Wu D, Ma Y, Fan S, Wang J, Gao N, Huang Z (2016) The crystal structure of Cpf1 in complex with CRISPR RNA. Nature 532:522\u0026ndash;526. https://doi.org/10.1038/nature17944\u003c/li\u003e\n\u003cli\u003eGao P, Yang H, Rajashankar KR, Huang Z, Patel DJ (2016) Type v CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Res 26:901\u0026ndash;913. https://doi.org/10.1038/cr.2016.88\u003c/li\u003e\n\u003cli\u003eHan X, Yang Y, Han X, Ryner JT, Ahmed EAH, Qi Y, Zhong G, Song G (2022) CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry. Plant Cell, Tissue Organ Cult 148:217\u0026ndash;229. https://doi.org/10.1007/s11240-021-02177-1\u003c/li\u003e\n\u003cli\u003eHernandez-Garcia A, Morales-Moreno MD, Vald\u0026eacute;s-Galindo EG, Jimenez-Nieto EP, Quezada A (2022) Diagnostics of COVID-19 Based on CRISPR\u0026ndash;Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update. Diagnostics 12:1434. https://doi.org/10.3390/diagnostics12061434\u003c/li\u003e\n\u003cli\u003eJinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (80- ) 337:816\u0026ndash;821. https://doi.org/10.1126/science.1225829\u003c/li\u003e\n\u003cli\u003eJirawannaporn S, Limothai U, Tachaboon S, Dinhuzen J, Kiatamornrak P, Chaisuriyong W, Bhumitrakul J, Mayuramart O, Payungporn S, Srisawat N (2022) Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR/Cas12a targeting lipL32. PLoS Negl Trop Dis 16:e0010112. https://doi.org/10.1371/journal.pntd.0010112\u003c/li\u003e\n\u003cli\u003eKhan S, Sallard E (2023) Current and Prospective Applications of CRISPR-Cas12a in Pluricellular Organisms. Mol Biotechnol 65:196\u0026ndash;205. https://doi.org/10.1007/s12033-022-00538-5\u003c/li\u003e\n\u003cli\u003eKim HK, Song M, Lee J, Menon AV, Jung S, Kang YM, Choi JW, Woo E, Koh HC, Nam JW, Kim H (2017) In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nat Methods 14:153\u0026ndash;159. https://doi.org/10.1038/nmeth.4104\u003c/li\u003e\n\u003cli\u003eKleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869\u0026ndash;874. https://doi.org/10.1038/nbt.3620\u003c/li\u003e\n\u003cli\u003eLee Yu H, Cao Y, Lu X, Hsing I-M (2021) Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity. Biosens Bioelectron 189:113382. https://doi.org/10.1016/j.bios.2021.113382\u003c/li\u003e\n\u003cli\u003eMartin L, Rostami S, Rajan R (2023) Optimized protocols for the characterization of Cas12a activities, 1st edn. Elsevier Inc.\u003c/li\u003e\n\u003cli\u003eMohanraju P, Oost J, Jinek M, Swarts D (2018) Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein. Bio-Protocol 8:1\u0026ndash;23. https://doi.org/10.21769/bioprotoc.2842\u003c/li\u003e\n\u003cli\u003eMorales-Moreno MD, Vald\u0026eacute;s-Galindo EG, Reza MM, Fiordelisio T, Peon J, Hernandez-Garcia A (2023) Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a. Cris J 6:116\u0026ndash;126. https://doi.org/10.1089/crispr.2022.0074\u003c/li\u003e\n\u003cli\u003ePaenkaew S, Jaito N, Pradit W, Chomdej S, Nganvongpanit K, Siengdee P, Buddhachat K (2023) RPA/CRISPR-cas12a as a specific, sensitive and rapid method for diagnosing Ehrlichia canis and Anaplasma platys in dogs in Thailand. Vet Res Commun 47:1601\u0026ndash;1613. https://doi.org/10.1007/s11259-023-10114-0\u003c/li\u003e\n\u003cli\u003ePaul B, Montoya G (2020) CRISPR-Cas12a: Functional overview and applications. Biomed J 43:8\u0026ndash;17. https://doi.org/10.1016/j.bj.2019.10.005\u003c/li\u003e\n\u003cli\u003ePouresmaeil M, Azizi-Dargahlou S (2023) Factors involved in heterologous expression of proteins in \u003cem\u003eE. coli\u003c/em\u003e host. Arch Microbiol 205:212. https://doi.org/10.1007/s00203-023-03541-9\u003c/li\u003e\n\u003cli\u003eRosano GL, Ceccarelli EA (2014) Recombinant protein expression in \u003cem\u003eEscherichia coli\u003c/em\u003e: advances and challenges. Front Microbiol 5. https://doi.org/10.3389/fmicb.2014.00172\u003c/li\u003e\n\u003cli\u003eShi Y, Fu X, Yin Y, Peng F, Yin X, Ke G, Zhang X (2021) CRISPR‐Cas12a System for Biosensing and Gene Regulation. Chem \u0026ndash; An Asian J 16:857\u0026ndash;867. https://doi.org/10.1002/asia.202100043\u003c/li\u003e\n\u003cli\u003eStrohkendl I, Saifuddin FA, Rybarski JR, Finkelstein IJ, Russell R (2018a) Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol Cell 71:816-824.e3. https://doi.org/10.1016/j.molcel.2018.06.043\u003c/li\u003e\n\u003cli\u003eStrohkendl I, Saifuddin FA, Rybarski JR, Finkelstein IJ, Russell R (2018b) Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a. Mol Cell 71:816-824.e3. https://doi.org/10.1016/j.molcel.2018.06.043\u003c/li\u003e\n\u003cli\u003eSwarts DC, Jinek M (2018) Cas9 versus Cas12a/Cpf1: Structure\u0026ndash;function comparisons and implications for genome editing. WIREs RNA 9. https://doi.org/10.1002/wrna.1481\u003c/li\u003e\n\u003cli\u003eWong MS, Wu S, Causey TB, Bennett GN, San K-Y (2008) Reduction of acetate accumulation in \u003cem\u003eEscherichia coli \u003c/em\u003ecultures for increased recombinant protein production. Metab Eng 10:97\u0026ndash;108. https://doi.org/10.1016/j.ymben.2007.10.003\u003c/li\u003e\n\u003cli\u003eYang H, Wang H, Wang F, Zhang K, Qu J, Guan J, Shen W, Cao Y, Xia Y, Chen X (2022) Efficient extracellular production of recombinant proteins in \u003cem\u003eE. coli\u003c/em\u003e via enhancing expression of dacA on the genome. J Ind Microbiol Biotechnol 49. https://doi.org/10.1093/jimb/kuac016\u003c/li\u003e\n\u003cli\u003eYang Y, Wang D, L\u0026uuml; P, Ma S, Chen K (2023) Research progress on nucleic acid detection and genome editing of CRISPR/Cas12 system. Mol Biol Rep 50:3723\u0026ndash;3738. https://doi.org/10.1007/s11033-023-08240-8\u003c/li\u003e\n\u003cli\u003eZarzhitsky S, Jiang A, E. Stanley E, H. Hecht M (2020) Harnessing synthetic biology to enhance heterologous protein expression. Protein Sci 29:1698\u0026ndash;1706. https://doi.org/10.1002/pro.3907\u003c/li\u003e\n\u003cli\u003eZetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, Van Der Oost J, Regev A, Koonin E V., Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759\u0026ndash;771. https://doi.org/10.1016/j.cell.2015.09.038\u003c/li\u003e\n\u003cli\u003eZhang Z-X, Nong F-T, Wang Y-Z, Yan C-X, Gu Y, Song P, Sun X-M (2022) Strategies for efficient production of recombinant proteins in \u003cem\u003eEscherichia coli\u003c/em\u003e: alleviating the host burden and enhancing protein activity. Microb Cell Fact 21:191. https://doi.org/10.1186/s12934-022-01917-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CRISPR-Cas12a, Protein biosynthesis, Recombinant production, Optimization, Lab-scale production","lastPublishedDoi":"10.21203/rs.3.rs-4535821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe CRISPR-Cas12a system is a groundbreaking tool that has seen an ample use for genome editing and diagnostics in biotechnology and biomedicine research labs. Despite its increasing use, there is a lack of studies on optimizing Cas12a protein production at lab-scale using straightforward protocols. This study aimed on enhancing the lab-scale recombinant production of \u003cem\u003eAcidaminococcus sp\u003c/em\u003e Cas12a protein (AsCas12a) in \u003cem\u003eE. coli\u003c/em\u003e. Through careful adjustments of simple parameters, the production of AsCas12a was remarkably increased. Optimized conditions involved using the BL21(DE3) strain, TB medium with 1% glucose, induction with 0.3 mM IPTG for at least 6\u0026ndash;9 h and incubation at 30\u0026deg;C. Notably, these conditions deviate from conventional production protocols for Cas12a and related proteins such as Cas9 from \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e. Upon combination of all optimized conditions bacterial production of AsCas12a improved\u0026thinsp;~\u0026thinsp;3 times, passing from 0.95 mg / mL of bacterial lysate volume, for non-optimized conditions, to 3.73 mg/mL in the optimal ones. The production yield of AsCas12a protein, after chromatographical purification increased\u0026thinsp;~\u0026thinsp;4.5 times, from 5.2 to 23.4 mg/L (culture volume) without compromising its functionality at all. The purified AsCas12a protein retained full activity for programmable \u003cem\u003ein vitro\u003c/em\u003e DNA \u003cem\u003ecis\u003c/em\u003e-cleavage and for collateral \u003cem\u003etrans\u003c/em\u003e-activity, which was used to detect the N gene from SARS-CoV-2. This optimized method offers an efficient and high-yield AsCas12a protein production using materials and conditions that are accessible to many research labs around the world.\u003c/p\u003e","manuscriptTitle":"Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 09:51:49","doi":"10.21203/rs.3.rs-4535821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revision","date":"2024-07-11T02:50:55+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-26T20:36:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-24T07:55:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-15T12:56:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"AMB Express","date":"2024-06-05T18:55:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2847007a-42cc-4525-bfa2-051805401ec6","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T06:04:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 09:51:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4535821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535821","identity":"rs-4535821","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}