Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference

Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 January 2025 V1 Latest version Share on Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference Authors : Neha Pandey , Chitra S. Misra , and Devashish Rath 0000-0002-8204-8440 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173648728.80649608/v1 Published Biochemical Journal Version of record Peer review timeline 204 views 99 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The structural and mechanistic complexity of Escherichia coli ’s type I CRISPR-Cas system compared to the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5, Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct Escherichia coli lineages, BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system and BL21, a natural mutant lacking the type I-E CRISPR-Cascade system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11 which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference. Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference Neha Pandey a b , Chitra S. Misra a , Devashish Rath a c * a Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai, India 400085. b Life Sciences, Mumbai University, Vidya Nagari, Kalina, Santacruz East, Mumbai, India 400098. c Homi Bhabha National Institute, Anushaktinagar, Mumbai, India 400094. * Correspondence: Devashish Rath, Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai-400085. India. Email: [email protected] phone: +91 22 25590796 Fax +91 22 25505326 Running Title: Cas11 is dispensable for in vivo CRISPR Cascade function. Abstract The structural and mechanistic complexity of Escherichia coli ’s type I CRISPR-Cas system compared to the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5, Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct Escherichia coli lineages, BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system and BL21, a natural mutant lacking the type I-E CRISPR-Cascade system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11 which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference. Keywords: CRISPR, Cascade, Cas11, type I-E, gene silencing, Escherichia coli 1. Introduction The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated) systems are adaptive immune mechanisms used by bacteria and archaea to defend against invasive nucleic acids such as bacteriophages and plasmids (1). These systems rely on signature Cas proteins along with a functional CRISPR RNA (crRNA) to mediate precise target recognition and its nucleolytic degradation. CRISPR/Cas systems are categorized into two major classes with several types and sub-types within each class. The class 1 systems have single Cas effector protein while class 2 systems, typically have multi-Cas protein effector complex. The types and sub-types are grouped based on their mode of action and presence of signature Cas proteins (2). Among the six types (I-VI), type I, type II, and type III systems, characterized by their distinctive signature proteins: Cas3, Cas9, and Cas10 respectively, were discovered early and were extensively studied (1). The type II CRISPR/Cas system, due to its relative structural simplicity, has been particularly well understood and is widely used for genome editing applications (3). The type I CRISPR/Cas system features a multiprotein complex known as Cascade (CRISPR-associated complex for antiviral defense) and a distinct nuclease, Cas3 that is responsible for the nucleolytic degradation of the target nucleic acid (4). Most of the knowledge of type I systems comes from studies of type I-E system in Escherichia coli ( E. coli ) (Fig.1A). The interaction of Cascade with foreign DNA begins with a search for a sequence called a protospacer-adjacent motif (PAM) (5). PAM recognition is followed by crRNA-guided search for complementarity between crRNA spacer and flanking DNA sequence (6,7). The binding of the spacer with the target triggers a conformational change that recruits Cas3. Cas3 recruitment by Cascade leads to degradation of the non-target strand in the 3’ to 5’ direction (4). The structure of the surveillance complex formed by Cascade has been solved (8–10). The type I-E Cascade complex with crRNA (crRNP) consists of the five Cas proteins (Cas8, Cas11, Cas7, Cas5 and Cas6) and a 61 nucleotide (nt) mature crRNA. The horse shoe-shaped structure of this complex has a molecular mass of 405 kDa, comprising one subunit of Cas8 (earlier Cse1), two subunits of Cas11 (earlier Cse2), six subunits of Cas7 and one subunit each of Cas5 and Cas6 (earlier Cas6e). The 61 nt crRNA is a highly structured molecule which is divided into a 32 nt spacer, an 8 nt-long handle at the 5′-end, followed by 21 nt-long repeat sequence with a terminal hairpin at 3′-end. In vitro studies have shown that the RNA endonuclease Cas6 situated at one end of the Cascade structure, converts pre-crRNA into mature crRNA, interacts with the downstream repeat-derived 3’ handle of the crRNA and remains associated with the rest of the complex for further action (11,12). The hexameric helical structure of Cas7 protects the crRNA while Cas8 is responsible for PAM (Protospacer adjacent motif) recognition and recruitment of Cas3. A dimer of two small subunits, Cas11, is thought to stabilize the formation of an R-loop structure and bind the displaced DNA strand (10). The crystal structure reveals the roles of each Cascade component in both annealing to the target site and maintaining structural stability. The type I-E system from E. coli has been exploited for several applications. Cascade along with Cas3 has been used for targeted DNA degradation, for sequence specific killing of microbes and for genome editing (13,14). In absence of Cas3, Cascade stably binds to target DNA without cleaving it, a property that has been used for programmable gene silencing. Engineering of crRNA to direct binding of Cascade to a promoter demonstrated efficient gene silencing (upto ∼1000- fold) while binding of the Cascade to within ORF led to a more modest silencing of the gene (15,16). This has provided a very powerful tool to study essential genes in E. coli and other bacteria (17–19). The type-I CRISPR system may offer several advantages over the type-II and type-V systems (10). Type-I Cascade recognizes a longer stretch of DNA (~32 bases) compared to other CRISPR systems (typically targeting. In addition, the processing of pre-RNA by Cas6 enables easier design of multiple targets. The multi-subunit protein complex also allows easy functionalization and programming for varied applications. Lastly, Cas3 being a processive enzyme, offers the possibility of creating larger deletions around the targeted locus (20). A minimal Cascade with lesser number of Cas proteins is likely to enhance the applicability of this system and make it easier to adapt it in heterologous systems. Here, towards the objective of constructing a minimal Cascade, a study was undertaken to decipher the dispensability of its components for gene silencing and plasmid interference in E. coli . We created different deletion variants of all five Cascade components, and assessed their functionality in terms of gene silencing and plasmid interference in two different E. coli genetic backgrounds. Our results show that Cas8, Cas5, Cas7 and Cas6 are indispensable for gene silencing and plasmid interference, however, engineered Cascade complex without Cas11 shows gene silencing and plasmid interference indicating functional redundancy. 2. Materials and Methods 2.1. Strains and culture media Most experiments were performed in the E. coli strain MLS367, which is a Δ cas3 derivative of E. coli K-12 BW25113 with an arabinose inducible T7 polymerase (ara::T7RNAP-tetA) gene integrated into its genome (16) and BL21DE3 or BL21AI strain that naturally lack Cas3 and Cascade. E. coli was cultured in Luria Bertani (LB) medium and grown with aeration at 37°C. The media was supplemented with kanamycin (25 μg/ml), ampicillin (100 μg/ml), and chloramphenicol (15 μg/ml), wherever required. To construct cas11 and cas6 mutants of MLS367, Keio collection (21) strains JW2726 ( Δ cas11::kan ) and JW2729 ( Δ cas6::kan ) were used as donors in P1 transduction (22) . MLS367 was used as a recipient and recombinants were selected on kanamycin. Δ cas11 and Δ cas6 derivatives of MLS367 were named as MLS366 and MLS368 respectively. Deletion of target genes ( cas11 or cas6 ) was verified by PCR amplification and sequencing (FigS1). Details of E. coli strains used in this study are given in Table S1. 2.2. Construction of recombinant plasmids For recombinant plasmid construction and transformation, standard methods as described were used (23). The recombinants were confirmed by restriction analysis and sequencing as required. Cascade operon was cloned in pRSF1b or pCDF1b at NcoI and NotI site and the resulting plasmids were named pRS-Cas or pCD-Cas respectively. Various deletion variants of the Cascade operon were generated from these two recombinants by designing inverse primers for the targeted deletion of individual genes. As an examples, for the deletion of cas8 (pRS-Δcas8), the inverse primers binding upstream and downstream of cas8 were used for amplification and the product was self-ligated to generate pRS-Δcas8. Similarly for generation of pRS-Δcas11, pRS-Δcas7, pRS-Δcas5 and pRS-Δcas6 primers binding upstream and downstream of the respective ORFs were used for inverse PCR and self-ligation of the products. The deletion of individual Cas gene was confirmed by diagnostic PCR using gene-specific and vector-specific primers and restriction enzyme digestion analysis of resultant plasmids. For expression of crRNA, CRISPR arrays containing the transcribed 53 bp of the leader sequence from the CRISPR1 array of E. coli K-12 MG1655 and a spacer flanked by two repeats were synthesized and cloned into the EcoRI and XbaI sites of pZE12Luc as reported earlier (16). For gene silencing experiments MLS367, BL21DE3, MLS368 and MLS366 cultures were serially transformed with pEH9 carrying GFP, crRNA expressing plasmids and pRS-Cascade or its deletion variants (pRS-Δcas6, pRS-Δcas5, pRS-Δcas7, pRS-Δcas11, pRS-Δcas8). Plasmids used in this study are listed in Table S2. Primers used in this study are given in Table S3. 2.3. Protein expression and analysis The Cascade complex and its deletion variants were expressed in E. coli BL21 (DE3) in LB broth with Kanamycin (25 µg/ml) or Streptomycin (15 µg/ml). After growing to OD600nm ∼0.5 at 37°C, expression was induced with 0.5 mM IPTG for 3 hours. Cells were harvested, lysed, and debris removed by centrifugation. The proteins were separated on SDS-PAGE gel and stained using Coomassie Brilliant blue. 2.4. Gene Silencing studies To evaluate the extent of gfp silencing in E. coli population, cultures grown overnight in LB medium were diluted 1:100 into 20 ml of fresh LB medium containing the appropriate antibiotics, 0.2% arabinose, and 0.5 mM IPTG to induce expression of crRNA and Cascade or its variants. The cultures were then incubated with aeration in 100 ml flasks at 37°C. Optical density (OD600) and fluorescence at 520 nm were measured at 5 hours post-inoculation during the exponential phase. For fluorescence measurements, 200 μl aliquots of the cultures were transferred to black Corning flat-bottom plates and read using an Infinite M200 Pro microplate reader (Tecan), (Excitation λ=480nm, Emission λ=520nm). Fluorescence was normalized to optical density (OD) values to account for variations in cell density. This normalization allowed for the assessment of relative fluorescence intensity. 2.5. Plasmid interference studies A 350 bp fragment derived from J gene of λ phage (λ350) cloned in pUC19 served as target for plasmid interference. E. coli (MLS367/BL21) cells, expressing crRNA 4XJ3 (targeting λ350) from pACYCduet1 plasmid, Cas3 from pRSF1b plasmid and Cascade, or its deletion variants on pCDF1b plasmid served as host. Cells expressing Cascade or its variants, crRNA and Cas3 were transformed with 100ng each of an empty vector control plasmid, pUC-19 or pUC19-λ350 test plasmid. Electroporation was done at 1400 Volts for high efficiency of transformation using Electroporator 2510 Eppendorf instrument. The cells were plated on LB-agar plates containing inducers, IPTG and arabinose for expression of the Cas3, Cascade and crRNA and ampicillin for selection of pUC19/pUC19- λ350 transformants. After overnight incubation, plasmid stability was evaluated by comparing the number of colony-forming units on the control plates to those on the test plates. 2.6. Microscopy The cells containing the silencing machinery were harvested after 5 hours of induction and washed twice with 1× phosphate-buffered saline (PBS). For fluorescence microscopy, the cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (0.5 μg/μL) to label the nuclei and FM 4-64FX (0.5 μg/μL), a membrane stain, for 30 minutes at room temperature in the dark. After staining, the cells were washed three times with 1× PBS and mounted onto a 1% agarose bed on a glass slide. Images were captured using a confocal microscope (Olympus IX83 FluoView 3000) with 100x magnification. The images were processed using cellSens imaging software, and representative images have been shown. 3. Results 3.1. Deletion of individual Cascade components alters the expression of other components from the cascade operon The regions containing CRISPR-Cas loci are the fastest evolving regions in the genome of bacteria (24). Further, horizontal gene transfer contributes to the rapid evolution of these loci. In order to confirm the status of CRISPR-Cas loci in our laboratory strains, PCR amplification of cas3 and Cascade operon which are key components of type I-E CRISPR-Cas system was carried out. Gene specific primers for the Cascade operon, and for cas3 , were used for amplification. No PCR products were observed for both cas3 and Cascade operon in E. coli BL21 (DE3) confirming their absence in this strain (Fig. S1). While E. coli K-12 strain BW25113 produced PCR products of expected sizes, 4.2 kb for cascade and 2.6 kb for cas3 , MLS367, a Δ cas3 derivative of BW25113 showed amplification of cascade operon but not for cas3 (Fig. S1). Previous reports indicate that the Cascade operon is repressed in BW25113 (25,26). We assessed whether residual expression of Cascade can support gene silencing. To test this, plasmids pZE12Luc encoding crRNA targeting the promoter(P1) or the ORF (T1) of gfp or a non-targeting scrambled crRNA, and pEH9 plasmid harbouring a Venus- gfp gene downstream of a PLtetO-1 promoter were transformed into MLS367 (16). Introduction of Cascade (pRS-Cas) in cells containing gfp targeting crRNA led to about 7 -fold decrease in GFP fluorescence compared to non-target control (Fig. 1B&C). However, upon introduction of empty vector pRSF1b, GFP fluorescence levels were similar to those obtained with non-target control (Fig. 1B&C). The results show that in absence of expression of Cascade from an external plasmid, no GFP silencing is observed in this strain indicating that the chromosomal Cascade operon is repressed and is unable to support gene silencing in presence of targeting crRNA. Fig. 1. Assessment of contribution of background expression of the Cascade system to GFP silencing in E. coli MLS367. Schematic representation of the Cascade operon in E. coli MLS367 (A). Fluorescence was monitored in MLS367 cells expressing GFP and crRNA (targeting gfp promoter, P1 or ORF, T1 or a non-targeting scrambled control, C) and carrying a plasmid encoding Cascade operon or empty vector (pRSF), through spot assays (A) and in broth cultures at 6 hours post-induction (B). Spot data is representative of experiments repeated 3 times (A). Data plotted is mean ± SEM of three independent experiments (B) ***p<0.001 by t test. The genes coding for all five components of Cascade, Cas8, Cas11, Cas7, Cas5 and Cas6 are transcribed from a single operon in the type I-E system of E. coli . Using pRS-Cas as a template, each of the five components of Cascade operon, were deleted separately. The expression of Cascade subunits in the deletion variants generated was checked by separation of total proteins on SDS-PAGE followed by Coomassie Brilliant blue staining. E. coli BL21 (DE3) was individually transformed with pRS-Cas, pRS-∆cas6, pRS-∆cas5, pRS-∆cas7, pRS-∆cas11and pRS-∆cas8 and the cells were induced with 0.5 mM IPTG. Total protein from these cells was analysed along with uninduced control on 13 % SDS-PAGE (Fig S2). In cells harbouring pRS-Cas, expression of Cas5 (25 kDa) and Cas6 (23 kDa) was not discernible, likely due to polar effect within the operon. In cells expressing full-length Cascade (pRS-Cas) or pRS-∆cas6 or pRS-∆cas5, expression of Cas8, Cas11 and Cas7 was apparent as protein bands of sizes 55 kDa, 17 kDa and 40 kDa respectively. Deletion of cas8 in plasmid-borne Cascade operon resulted in expression of Cas5 and Cas6 in addition to Cas11 and Cas7 albeit at lower levels. In case of cas11 deletion, all four remaining subunits were expressed with Cas8 and Cas7 showing higher levels in comparison to Cas5 and Cas6. Overall, the analysis showed that Cas5 and Cas6 are expressed at low levels from the Cascade operon (Fig. S2). Their expression levels improved with the deletion of a preceding gene in the operon. 3.2. Essentiality of Cascade components for gene silencing To assess the functional essentiality of Cascade components, variants lacking one of the Cascade components were tested in gene silencing assays and compared with the full-length Cascade in MLS367 an E. coli K-12 strain, using a protocol described earlier (16). MLS367 cells carrying gfp reporter under a constitutive promoter on plasmid pEH9 were transformed with plasmids expressing promoter-specific (P1, P2) or ORF specific (NT1, T1) crRNAs (Fig. 2A). Cascade or Cascade deletion variants lacking any one of the 5 components at a time were introduced into these strains. Gene silencing was induced and GFP expression was monitored on agar plates as well as broth. In cells expressing Cascade, maximum silencing was obtained with crRNA-P1 and crRNA-P2 (6.5-fold each), followed by crRNA-T1 (4.1-fold) and crRNA-NT1 (1.9-fold) (Fig. 2B & C). Deletion variants lacking cas8 or cas7 or cas5 did not show silencing with any of the crRNAs suggesting that these subunits of Cascade are essential for gene silencing. Deletion of cas6 or cas11 caused decreased fluorescence with about half the level of silencing obtained with Cascade (Fig. 2B & C). This showed that in E. coli K-12 strain, Cascade lacking Cas11 or Cas6 could still give effective gene silencing suggesting that these Cas subunits could be dispensable for gene silencing application. The above experiments were performed in E. coli BL21 (DE3), a strain that lacks the genomic Cascade operon. With Cascade and crRNA-P1 or crRNA-P2, a 10-fold gfp silencing was obtained while crRNA-T1 and crRNA-NT1 could bring about 6- and 2-fold silencing of the reporter respectively (Fig. 3A & B). In this genetic background, expression of Cascade variants lacking Cas8 or Cas7 or Cas5 did not show gene silencing. Cascade lacking the Cas6 too did not show gene silencing suggesting that all the four subunits of Cascade were essential (Fig. 3A & B). Interestingly, in this strain, Cascade variant without the Cas11 subunit displayed about half the level of gene silencing seen with complete Cascade (Fig. 3A & B), as was observed with MLS367. While the results with Cas6 remained inconclusive, identical results in two independent genetic backgrounds show that Cas11 is dispensable for Cascade mediated silencing of gene expression. Fig. 2. Effect of deletion of individual Cascade components on gfp silencing in E. coli MLS367. Binding positions of crRNAs in the promoter and the ORF of gfp (A). Fluorescence was monitored in cells expressing GFP and crRNAs (targeting gfp promoter, P1 and P2 or ORF, NT1 and T1 or a non-targeting scrambled control, C) and Cascade or its variants was monitored by spot assay (B) and in broth at 6hr post induction (C). Spot data is representative of experiments repeated 3 times (B). Data plotted is mean ± SEM of three independent experiments (C). *p<0.05, **p<0.01, ***p<0.001 by t test. Fig. 3. Effect of deletion of individual Cascade components on gfp silencing in E. coli BL21(DE3). Binding positions of crRNAs in the promoter and the ORF of gfp (A). Fluorescence was monitored in cells expressing GFP and crRNAs (targeting gfp promoter, P1 and P2 or ORF, NT1 and T1 or a non-targeting scrambled control, C) and Cascade or its variants was monitored by spot assay (B) and in broth at 6hr post induction (C). Spot data is representative of experiments repeated 3 times (B). Data plotted is mean ± SEM of three independent experiments (C). *p<0.05, **p<0.01, ***p<0.001 by t test. 3.3. Cas6 is required for Cascade-based gene silencing The observed redundancy of Cas6 for gene silencing in MLS367 a K-12 strain of E. coli was intriguing. We hypothesized that it might be due to residual expression of Cas6 in an otherwise repressed Cascade operon in this strain. To ascertain this, we constructed knockouts of cas11 and cas6 of MLS367 by transduction using the corresponding mutants from the Keio collection (21). Gene silencing was analyzed through in-trans complementation with plasmid borne Cascade or its variants. Mutant cells lacking chromosomal copy of cas11 (MLS366) but expressing cas11 deletion variant of Cascade in-trans, continued to show gene silencing albeit at a lower level than obtained with complete Cascade (Fig 4A). For instance, upon using crRNA-P1, an approximately 10-fold reduction in gene silencing was observed with full-length Cascade, but a 4-fold silencing was sustained upon providing Cascade lacking Cas11 (Fig 4A). Fig. 4. Effect of chromosomal deletion of cas6 and cas11 on gfp gene silencing in K-12 background of E. coli . Gene silencing with Cascade and ∆ cas11 Cascade expressed from plasmid in MLS367 and MLS366 (∆ cas11 MLS367) strain (A). Gene silencing with Cascade and ∆ cas6 Cascade expressed from plasmid in MLS367 and MLS368 (∆ cas6 MLS367) strain (B). C: non-targeting crRNA, P1 or T1: targeting crRNA. Data plotted is mean ± SEM of three independent experiments. **p<0.01, ***p<0.0001 by t test. In contrast, in a strain lacking the chromosomal copy of cas6 (MLS368), gene silencing was not observed when complemented with a Cascade lacking Cas6 (Fig. 4B). However, when such cells were complemented with the full Cascade, a 6.7-fold GFP silencing was recorded (Fig. 4B). This clearly indicated leaky expression of cas6 from the chromosomal Cascade operon in MLS367. Together these results suggest that while Cas11 protein augments efficiency, it is not essential for the gene silencing function of Cascade complex but Cas6 is needed for its functional activity. 3.4. Minimal Cascade lacking Cas11 can silence native gene in E. coli In order to assess and quantify gene silencing, heterologous expression of GFP from a plasmid was used as a convenient reporter in previous experiments. To show that the minimal Cascade minus the Cas11 subunit can be used to silence a native gene on the genome, we targeted racR . Earlier reports showed that racR was an essential gene within the rac prophage of E. coli K-12 strains and its deletion made cells unviable (17). Further, Bindal et al (2017) showed that silencing of racR in E. coli K-12 MG1655 strain significantly retarded growth and altered cell morphology, leading to formation of long filamentous cells (17). Silencing of racR in MLS366 using a targeting crRNA, either with full-length Cascade or minimal Cascade, reduced growth compared to cells expressing a nontargeting crRNA (Fig. 5A). The cells were visualized by microscopy after induction of silencing. Control cells expressing a nontargeting crRNA displayed normal size and shape. However, cells expressing Cascade or Cascade lacking Cas11 and a racR targeting crRNA showed striking morphological changes (Fig. 5B) such as extensive filamentation and an increase in cell diameter confirming silencing of racR expression in these cells. The results confirmed that Cas11 subunit is dispensable and also that Cascade lacking this subunit can be effectively utilized for gene silencing applications. Fig. 5. Effect of racR silencing with Cascade and ∆cas11 Cascade. Growth defect (A) and morphological defect (B) resulting from RacR depletion. Growth was monitored in MLS366 ( ∆cas11 MLS367) cells expressing Cascade or ∆cas11 Cascade and racR -targeting crRNA (T) or non-targeting crRNA (NT). Cells were analyzed using confocal microscopy at 100x magnification 5 hr after the induction of transcriptional silencing. Data plotted is mean ± SEM of three independent experiments. 3.5. Cas11 is dispensable for CRISPR-Cascade-mediated plasmid interference Cascade is the main effector in the type IE CRISPR-Cas system that functions in bacterial immunity against mobile genetic elements (MGE). The spacer sequence of the crRNA is utilised by Cascade for recognising the incoming MGE through RNA-DNA complementary base pairing. This leads to simultaneous recruitment of a nuclease Cas3 which eventually degrades the MGE in a processive manner, a process referred to as interference. Plasmid interference was monitored in E. coli cells expressing both Cas3 and Cascade or its deletion variants on plasmids. For antibiotic selection compatibility reasons, Cascade and its deletion variants used in gene silencing experiments could not be used for interference studies. Hence, Cascade or its deletion variants described earlier were sub-cloned in pCDF1b under a T7 promoter. The cells expressing Cascade deletion variants were evaluated by SDS-PAGE analysis of E. coli . Cells harbouring different variants of Cascade in pCDF1b showed that the protein profile was similar to the profile obtained with pRSF-derivatives and broadly indicated expression of Cascade subunits except the one deleted from the respective construct (data not shown). The stability of target plasmid pUC-λ350 (plasmid containing 350 base pair of λ) or control plasmid pUC19 upon transformation was monitored in cells expressing Cas3, 4XJ3 (CRISPR array containing four copies of the J3 spacer targeting λ350), crRNA and Cascade or its deletion variants. The scheme depicting experimental design of interference assay is shown in Fig. 6A. The recovery of the pUC19-λ350 (target) transformants was compared with pUC19 (non-target) transformants to determine the effect of plasmid targeting by Cascade and the deletion variants. pACYCDuet-1 carrying a crRNA against a scrambled sequence was used as a control to show that plasmid interference was dependent on the presence of targeting crRNA. The interference assays were carried out in both E. coli K-12 (MLS367) and B strain (BL21) backgrounds (Fig. 6B and C). Results showed that transformants for pUC-λ350 could not be recovered in the presence of full-length Cascade and the targeting 4XJ3 test crRNA in both E. coli K-12 (BW25113) and B strain (BL21) backgrounds (Fig. 6B and C), while good transformation efficiency was obtained with pUC19 vector alone. However, the number of transformants recovered were similar upon transforming pUC19 or pUC-λ350 in cells expressing a scrambled non-targeting control crRNA. This showed that the plasmid interference was equally functional in both the genetic backgrounds. With Cascade variants, results were similar to those obtained with gene silencing assays. Plasmid interference did not take place upon deletion of cas8, cas7 or cas5 in either of the backgrounds suggesting that these three subunits of Cascade are essential for interference. Cascade without Cas6 affected plasmid stability in K-12 background (Fig 6B) but this was not observed in B strain background (Fig 6C). Interestingly, both strains of E. coli showed low plasmid stability in the presence of minimal Cascade lacking Cas11 suggesting that Cascade lacking Cas11 could still support interference, In BW25113, there was a 4-fold reduction in the number of transformants in absence of Cas11, when the cells are transformed with the target plasmid, whereas in BL21, there was only a 2-fold reduction. In summary, these results demonstrate that the Cascade complex effectively targets and eliminates plasmids in the K-12 background, even in the absence of Cas11 or Cas6. However, in the B strain background, Cas6 is essential while Cas11 is dispensable. Experiments to confirm dispensability of cas6 in the K-12 strain could not be performed due to inability to generate a mutant with an antibiotic cassette insertion that was unique from those being engaged for selection of the rest of the four plasmids required for interference. Fig. 6. Plasmid interference with Cascade and Cascade variants. A schematic for the plasmid interference assay is shown (A). E. coli MLS367 (B) and E. coli BL21 (C) cells expressing Cas3, Cascade or Cascade deletion variants and targeting crRNA (Test) or a scrambled non targeting crRNA (Control) were transformed with 100 ng of each of the plasmids pUC19-λ350 or pUC19. Transformants recovered on ampicillin plates were enumerated. Relative transformation efficiency with respect to transformants obtained for pUC-19 control in cells expressing targeting crRNA is plotted. 4. Discussion The sea-horse shaped structure of the type I-E CRISPR system from E. coli has been studied in detail and characterized extensively (27–29). The function of each of the subunits is well annotated and experimentally verified. The crystal structure of the E. coli Cascade complex, resolved at 3.05 Å, showed that six Cas7 proteins, along with Cas5 and Cas6, create a tightly packed outer layer, while the inner layer consists of a Cas8 and Cas11 dimer. The structure revealed that the 61-nucleotide crRNA spans the entire complex, interacting with all six Cas7 subunits. The inner layer connects to the outer layer primarily through Cas8-Cas5 interactions, supplemented by multiple, relatively weak contact points between Cas7 and the lateral regions of the Cas11 dimer that occupies the ‘belly’ region on the Cascade complex(9). There has been a plethora of in vitro studies on Cascade components and their interaction with crRNA and/or target DNA. Early studies found that the absence of Cas8 and Cas11 did not impact the structural stability of the remaining subunits in the complex, nor did it interfere with the formation of mature crRNA (8). A recent study demonstrated, in vitro, that the Cas5-Cas7 scaffold is sufficient for PAM independent DNA targeting when using a mature crRNA. The study also showed R-loop formation and DNA cleavage in absence of Cas11 but at much lower efficiency compared to full-length Cascade (30). Structure-based studies have shown that Cas11 interacts with the non-target strand and locks it. This is hypothesized to aid stabilization of R-loop (28) and consequent conformational change in the Cascade complex to enable Cas3 recruitment for target cleavage. We hypothesized that as interference requires additional steps over CRISPRi such as recruitment of Cas3, stabilization of R-loop and activation of nuclease, requirement of Cas components could be different for DNA cleavage or at least more stringent than CRISPRi. Dispensability of Cas11 for DNA cleavage shown in our study indicates that role of Cas11 in stabilization of R-loop may not be crucial or maybe overestimated for Cas3-mediated cleavage in vivo. There have been few studies in vivo that have looked at minimal Cascade in type I-E system. This is perhaps the first study to assess gene silencing with Cascade variants. Our work shows that Cas11 is dispensable for not only Cascade-mediated gene silencing that involves binding of the surveillance complex to a specific DNA molecule but also for plasmid interference which involves in addition to DNA binding, Cas3 recruitment in vivo and DNA cleavage. Jore et al., expressed single component deletion variants of Cascade from plasmid in BL21(DE3) which is identical to our study and found no lambda phage interference in any of the variants concluding that all components are essential (8). Our results are in variance and we show that plasmid interference is supported by Cas11 deletion variant of Cascade. These apparently different outcomes could be due to different genetic elements used for expression of type IE CRISPR system components or differences in the requirement for plasmid vs phage interference. While Cascade type I-E encodes a distinct Cas11 subunit, the presence of Cas11 in type I-B, I-C, and I-D went unnoticed until recently. The type I-D Cascade from Synechocystis sp. PCC 6803 had an alternative internal translational initiation site within cas10d that lead to the expression of the small subunit, Cas11d (31). DNA binding was drastically reduced in absence of the Cas11d subunit. The minimal type 1-C system from Neisseria lactamica had a ‘hidden’ cas11 sequence within the cas8 sequence and was required for genome editing in eukaryotes (32). Type I-C system from Desulfovibrio vulgaris , similarly had a ‘hidden’ cas11 which was a strict requirement for successful genome editing (33). The type I-B system from Synechocystis also carries a hidden cas11 that significantly boosted editing efficiency when supplied separately (32). The type I-F CRISPR system, on the other hand, lacks Cas11. Protection against Lambda phage was demonstrated using Type 1-Fv of this minimal version (34). The dispensability of Cas11 for a functional Cascade therefore appears to vary for each subtype of Cascade. We also demonstrated that the minimal I-E CRISPR-Cas system from E. coli can effectively silence GFP gene expression and interfere with plasmid transformation, even in the absence of Cas6 activity in the BW5113 strain. In natural conditions, the expression of the Cascade operon in E. coli is known to be repressed in this strain (26,35). However, indispensability of Cas6 in the BL21 strain which is a natural mutant for the Cascade locus shows that Cascade complex indeed needs this subunit for DNA binding and cleavage when a pre-crRNA is provided. The dispensability of Cas6 in BW5113 strain is puzzling, especially since any background expression was ruled out by checking for silencing of GFP gene from the chromosomal Cascade alleles. One possibility could be that the Cascade locus is expressed from the chromosome at very low levels in BW5113 that is insufficient for formation of an active Cascade complex. Since Cas6 is employed only in a single copy in the Cascade complex, this level of expression might have been sufficient to engage with the rest of Cascade subunits expressed in excess from the plasmid to bring about GFP silencing. Why this was not true for Cas8 and Cas5, which are also engaged in single copy in the Cascade complex is not quite clear. Cas6, a component of the Cascade complex and an endoribonuclease, typically generates unit-sized crRNA from the pre-crRNA precursor transcript and is essential for CRISPR-Cas function. It was also shown that when mature unit-sized crRNAs are provided through transcription termination, the Cascade complex could bring about bacteriophage interference even in absence of Cas6 in E. coli (12). In the type I-B system from Haloferax also, it has been shown that Cas6 is not essential for crRNA binding to the Cascade complex or for target recognition (36). Removal of all but one nucleotide of the 3′ handle of crRNA was shown to still bind Type1 Cascade in vitro (37) as well as in vivo (36). Further, Cascade lacking Cas6 could still recognize the target when provided with a mature crRNA lacking the 3’ handle in vitro. Our study has shown the dispensability of the Cas11 protein when a full pre-crRNA was provided for DNA binding and cleavage in vivo. It remains to be seen if a minimal Cascade without Cas6 and Cas11 can still form a stable complex with a mature crRNA lacking the 3’ handle for application in genome editing and silencing. Author Credits Neha Pandey: Investigation; Writing – original draft. Chitra S. Misra: Data curation; Supervision; Writing – original draft; Writing – review and editing. Devashish Rath: Conceptualization; Supervision; Writing – review and editing. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Westra ER, Pul Ü, Heidrich N, Jore MM, Lundgren M, Stratmann T, et al. H‐NS‐mediated repression of CRISPR‐based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol Microbiol. 2010 Sep 14;77(6):1380–93. 36. Maier LK, Stachler AE, Saunders SJ, Backofen R, Marchfelder A. An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein. Journal of Biological Chemistry. 2015 Feb;290(7):4192–201. 37. Beloglazova N, Kuznedelov K, Flick R, Datsenko KA, Brown G, Popovic A, et al. CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli. Nucleic Acids Res. 2015 Jan 9;43(1):530–43. Figure Legends Fig. 1. Assessment of contribution of background expression of the Cascade system to GFP silencing in E. coli MLS367. Schematic representation of the Cascade operon in E. coli MLS367 (A). Fluorescence was monitored in MLS367 cells expressing GFP and crRNA (targeting gfp promoter, P1 or ORF, T1 or a non-targeting scrambled control, C) and carrying a plasmid encoding Cascade operon or empty vector (pRSF), through spot assays (A) and in broth cultures at 6 hours post-induction (B). Spot data is representative of experiments repeated 3 times (A). Data plotted is mean ± SEM of three independent experiments (B) ***p<0.001 by t test. Fig. 2. Effect of deletion of individual Cascade components on gfp silencing in E. coli MLS367. Binding positions of crRNAs in the promoter and the ORF of gfp (A). Fluorescence was monitored in cells expressing GFP and crRNAs (targeting gfp promoter, P1 and P2 or ORF, NT1 and T1 or a non-targeting scrambled control, C) and Cascade or its variants was monitored by spot assay (B) and in broth at 6hr post induction (C). Spot data is representative of experiments repeated 3 times (B). Data plotted is mean ± SEM of three independent experiments (C). *p<0.05, **p<0.01, ***p<0.001 by t test. Fig. 3. Effect of deletion of individual Cascade components on gfp silencing in E. coli BL21(DE3). Binding positions of crRNAs in the promoter and the ORF of gfp (A). Fluorescence was monitored in cells expressing GFP and crRNAs (targeting gfp promoter, P1 and P2 or ORF, NT1 and T1 or a non-targeting scrambled control, C) and Cascade or its variants was monitored by spot assay (B) and in broth at 6hr post induction (C). Spot data is representative of experiments repeated 3 times (B). Data plotted is mean ± SEM of three independent experiments (C). *p<0.05, **p<0.01, ***p<0.001 by t test. Fig. 4. Effect of chromosomal deletion of cas6 and cas11 on gfp gene silencing in K-12 background of E. coli . Gene silencing with Cascade and ∆ cas11 Cascade expressed from plasmid in MLS367 and MLS366 (∆ cas11 MLS367) strain (A). Gene silencing with Cascade and ∆ cas6 Cascade expressed from plasmid in MLS367 and MLS368 (∆ cas6 MLS367) strain (B). C: non-targeting crRNA, P1 or T1: targeting crRNA. Data plotted is mean ± SEM of three independent experiments. **p<0.01, ***p<0.0001 by t test. Fig. 5. Effect of racR silencing with Cascade and ∆cas11 Cascade. Growth defect (A) and morphological defect (B) resulting from RacR depletion. Growth was monitored in MLS366 ( ∆cas11 MLS367) cells expressing Cascade or ∆cas11 Cascade and racR -targeting crRNA (T) or non-targeting crRNA (NT). Cells were analyzed using confocal microscopy at 100x magnification 5 hr after the induction of transcriptional silencing. Data plotted is mean ± SEM of three independent experiments. Fig. 6. Plasmid interference with Cascade and Cascade variants. A schematic for the plasmid interference assay is shown (A). E. coli MLS367 (B) and E. coli BL21 (C) cells expressing Cas3, Cascade or Cascade deletion variants and targeting crRNA (Test) or a scrambled non targeting crRNA (Control) were transformed with 100 ng of each of the plasmids pUC19-λ350 or pUC19. Transformants recovered on ampicillin plates were enumerated. Relative transformation efficiency with respect to transformants obtained for pUC-19 control in cells expressing targeting crRNA is plotted. Information & Authors Information Version history V1 Version 1 10 January 2025 Peer review timeline Published Biochemical Journal Version of Record 11 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords escherichia coli cas11 cascade crispr type i-e Authors Affiliations Neha Pandey Bhabha Atomic Research Centre View all articles by this author Chitra S. Misra Bhabha Atomic Research Centre View all articles by this author Devashish Rath 0000-0002-8204-8440 [email protected] Bhabha Atomic Research Centre View all articles by this author Metrics & Citations Metrics Article Usage 204 views 99 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Neha Pandey, Chitra S. Misra, Devashish Rath. Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference. Authorea . 10 January 2025. DOI: https://doi.org/10.22541/au.173648728.80649608/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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