MosTI single-copy transgene insertion in C. elegans using heat-shock inducible Cas9 expression

MosTI single-copy transgene insertion in C. elegans using heat-shock inducible Cas9 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article MosTI single-copy transgene insertion in C. elegans using heat-shock inducible Cas9 expression Sonia El Mouridi, Christian Frøkjær-Jensen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2053151/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Precise expression of transgenes in C. elegans can be used to understand gene regulation, control cells, or as a starting point for genetic screens. Insertion of single-copy transgenes into well-defined safe-harbor locations is useful when consistent expression levels are required ( e.g. , to compare expression from different transgenes) or when expression is desired in germ cells. Here, we describe a detailed protocol for inserting single-copy transgenes using Modular Safe-harbor Transgene Insertion (MosTI) and a heat-shock inducible Cas9 expressed from a co-injected plasmid. Inducible Cas9 expression has the advantage of requiring few injections, and many independent single-copy insertions can be generated from a single array line. Biotechnology and Bioengineering C. elegans genetic engineering single-copy transgene Cas9 Figures Figure 1 Introduction The ease of generating transgenic C. elegan s is a significant strength of this genetic model system. The simple injection of DNA fragments (linear or circular) generates heritable extra-chromosomal arrays containing many DNA copies at high frequency (Stinchcomb et al., 1985). Optimized injection conditions ( e.g. , DNA concentration and the identification of a genetic selection marker)(Mello et al., 1991) have made transgenesis routine in many C. elegans laboratories. Extra-chromosomal arrays have some clear advantages: somatic transgene expression is frequently high due to transgene copy number (approx. 100 plasmids in an array), the genome is unaltered, and arrays are easy to lose with no requirement for genetic crosses due to mitotic loss in cell divisions. The disadvantages of arrays include the requirement for continuous selection, highly variable expression, and transgenes that are often silenced in the germline (Kelly et al., 1997). It is possible to partially mitigate germline array silencing by adding complex DNA to the injection mix (Kelly et al., 1997) or by linearizing plasmids and including a class of non-coding DNA structures (PATCs) (Aljohani et al., 2020; Fire et al., 2006; Frøkjær-Jensen, 2021; Frøkjær-Jensen et al., 2016), but these manipulations are somewhat cumbersome. Genomic insertion of low or single-copy transgenes can overcome many of the limitations of arrays. Random low-copy insertions can be generated by biolistic transformation using genetic selection markers (Praitis et al., 2001) or antibiotic selection (Radman et al., 2013; Semple et al., 2012). Some transgenic animals generated by biolistic transformation show stable germline expression (Merritt et al., 2008; Praitis et al., 2001), but transgene insertions are inherently mutagenic with occasional large-scale chromosome duplications, and transgenes are often truncated (Tyson et al., 2018). Transgenes can also be inserted at random locations by transposition with an optimized Mos1 transposon (Frøkjær-Jensen, 2015; Frøkjær-Jensen et al., 2014), which has the advantage that the exact insertion sites are easily determined (Boulin and Bessereau, 2007). However, the random nature of the transgene insertion site results in position effect variegation evidenced by variable somatic expression and frequent germline silencing(Frøkjær-Jensen et al., 2016). For these reasons, we and others have developed targeted-single copy transgene insertion methods based on homologous repair at double-strand DNA breaks (DSBs). The initial methods for modifying the genome were based on generating DSBs at a well-defined location using a native (Barrett et al., 2004) or exogenous Mos1 transposons (Bessereau et al., 2001; Frøkjær-Jensen et al., 2008, 2010; Robert and Bessereau, 2007). Using an extensive collection of strains with mapped Mos1 insertions (Vallin et al., 2012), it was possible to identify safe-harbor insertion sites, where transgenes were expressed at native levels and in the germline from the pie-1 (Frøkjær-Jensen et al., 2008) or mex-5 (Zeiser et al., 2011) promoters. Mos1-mediated single-copy transgene insertions (MosSCI) could be generated at a reasonably high frequency using genetic (Frøkjær-Jensen et al., 2008, 2012) and antibiotic (Giordano-Santini et al., 2010; Semple et al., 2010) selection markers. Later MosSCI iterations used similar selection strategies but generated DSBs using CRISPR/Cas9 (Dickinson and Goldstein, 2016; Dickinson et al., 2013; Frøkjær-Jensen, 2013). More recently, methods to insert single-copy transgenes based on recombination-mediated cassette exchange have also been developed (Nonet, 2021, 2020). One bottleneck for these targeted insertion methods is to distinguish between positive selection from arrays and single-copy inserts ( e.g. , unc-119 rescue could be conferred by the intermediate extra-chromosomal arrays used to generate single-copy insertions). Selecting single-copy insertions is facilitated by co-injecting a single muscle-expressed fluorescent marker (El Mouridi et al., 2020) or co-injection negative array selection markers, such as inducible expression of a toxin (Frøkjær-Jensen et al., 2012) or drug-inducible paralysis (El Mouridi et al., 2021). However, both approaches require significant screening or additional manipulations, respectively. To facilitate the identification of single-copy insertions, we and others have recently developed methods that rely on "split" selection markers (El Mouridi et al., 2022; Stevenson et al., 2020). With these methods, selection markers are split between the transgene vector and the safe-harbor landing site: the selection marker is only reconstituted and functional when a transgene is correctly insertion, which significantly facilitates screening for insertions ( Figure 1 ). The split selection markers include antibiotic (hygromycin) (Stevenson et al., 2020), genetic ( unc-119 ), and visual markers (GFP fluorescence) (El Mouridi et al., 2022; Stevenson et al., 2020). Both split-selection methods rely on CRISPR/Cas9 expression to induce DSBs, but the methods differ in several respects that favor different experimental setups. Stevenson et al .(2020) developed a split antibiotic selection marker (hygromycin) and focused on generating transgene insertions based on in situ assembly from several PCR fragments. Their method has the advantage of injecting into wild-type animals and an assembly method that requires no cloning, which, in principle, can be multiplexed. The disadvantages are that insertion frequencies are relatively low, homozygous animals are not as easy to generate using antibiotic selection, and the in situ assemblies are error-prone. We developed MosTI with the intention of incorporating several incremental single-copy transgenesis improvements, including split-selection markers, developed across many laboratories. We aimed to generate a set of standardized reagents, with several validated safe-harbor landing sites permissive for germline expression, an increased number of split selection markers ( unc-119 and Pmlc-2::gfp ), and a collection of target plasmids that contain common regulatory elements. Importantly, MosTI is compatible with single-copy transgene insertion and targeted array integration. Furthermore, the safe-harbor landing sites are modular and can be converted from one selection to another for added flexibility. One key feature of MosTI is the ability to generate DSBs with Cas9 using several strategies: constitutive expression of a PATC-rich Cas9, heat-shock inducible Cas9 expression from linearized transgenes, and two-step injections of Cas9 protein complexed with crRNA/tracrRNAs. Two pairs of divergently transcribed heat-shock promoters (Russnak and Candido, 1985), hsp-16.48 / hsp-16.1 and hsp-16.41 / hsp-16.2 , are commonly used for heat-inducible expression of transgenes in C. elegans (Stringham et al., 1992). Extra-chromosomal arrays carrying transgenes expressed under any one of the hsp promoters can drive inducible expression in most somatic cells at high levels after a short heat shock (1-2 hours at 33°C), but no expression was observed in the germ line or early embryo. Similarly, GFP expression from a single-copy hsp transgene is bright in somatic cells but very dim in germ cells (Zeiser et al., 2011). The mechanisms that limit heat-shock expression in germ cells are not well-defined, but modified transgenes with PATCs (Aljohani et al., 2020) and an improved extended heat-shock protocol (Nonet, 2021) can improve inducible Cas9 expression (El Mouridi et al., 2022). Here, we describe a detailed protocol for generating single-copy transgene insertions using heat-shock inducible germline expression of Cas9 from arrays and selecting insertion events by propagating animals over generations. Experimental Design Brief overview Inserting a single-copy transgene by MosTI consists of several steps ( Figure 1 ): (1) Transgene cloning . Cloning transgenes into the appropriate target vector. The target vectors are specific to the split selection marker ( i.e. , there are different vectors for unc-119 and Pmlc-2::gfp ). However, using a given selection marker, the target vectors are universal for all safe-harbor landing sites. (2) Micro-injections and identifying MosTI insertions . This step requires injecting plasmids into the appropriate MosTI strain using standard techniques, selecting for arrays, and screening for single-copy transgene insertion. (3) Insert validation (optional but recommended) . The final steps include removal of the selection marker, validating the integrity of the transgene insertion, and genetic outcrossing. Necessary expertise Fundamental molecular biology expertise is required to clone transgenes into targeting vectors, e.g. , restriction enzyme cloning, Golden-gate cloning (Engler et al., 2008), or Gibson assembly (Gibson et al., 2009). Alternatively, transgenes can be generated directly by gene synthesis. We have deposited a MosTI target vector for split unc-119 selection (pSEM246) at Twist Biosciences, which is available for custom vector gene synthesis by email request to the corresponding author. Basic expertise in C. elegans microinjection is required. The protocol was optimized to minimize the number of injections required by allowing inducible germline Cas9 expression from arrays; thus, only a few transgenic lines are necessary for generating several independent MosTI insertions. Limitations Transgenes can serve essential experimental functions but may only approximate how endogenous genes function. For example, regulatory elements may be position-dependent, and the usage of selection markers could have subtle effects on animals that are difficult to identify. Also, C. elegans have many competing small RNA and non-coding DNA pathways that enable germ cells to distinguish between endogenous and foreign DNA, such as transgenes (Frøkjær-Jensen, 2019). We suggest that experimenters use common sense and critically evaluate their results, preferably by having separate lines of experiments that do not depend on transgenes to support or refute observations. Materials Biological materials Required plasmids MosTI target vectors: pSEM246 ( unc-119 selection) or pSEM272 (P mlc-2 :: gfp selection) Co-injection plasmids: pSEM231 (P mlc-1 :: gfp ), pSEM233 (P mlc-1 :: tagRFP ), and pCFJ782 (HygroR). Cas9 plasmid: pMDJ231 (P hsp-16.41 :: cas9::gpd-2::tagRFP ). sgRNA plasmids: pSEM318 (split unc-119 ) or pSEM255 (split P mlc-2 :: gfp ) 1 kb Plus DNA ladder (500 ng/ul, no dye, Invitrogen #10787018) Note: see Table 3 for all MosTI plasmids and their Addgene IDs. Some target vectors with common promoters and fluorophores may facilitate generating transgenes following common design rules (Nance and Frøkjær-Jensen, 2019). MosTI injection strains (see Table 2 ). Strains are distributed by the Caenorhabditis elegans Genetics Center (CGC). OP50 and HB101 bacterial cultures. These are distributed by the Caenorhabditis elegans Genetics Center (CGC). Reagents Milli-Q water or distilled water. Nematode Growth Medium (NGM). Standard recipe as described in Stiernagle (2006). 6 cm vented Petri dishes for NGM plates. Standard molecular biology reagents ( e.g. , restriction enzymes to generate and validate transgenes). ApaLI (NEB cat. no. R0507S). PureLink HQ mini plasmid purification kit (cat. no. K2100-01). Note: Higher purity DNA can improve single-copy insertion frequencies (Frøkjær-Jensen, unpub. obs.), but you can use any commercial miniprep kit. Monarch PCR & DNA Cleanup Kit (NEB cat. no. T1030S). Hygromycin B (Gold Biotechnology, cat. no. H270-1). 500 mL single-use sterile vacuum filter ( e.g. , Fisher Scientific 09-741-20200). 50 mL conical centrifugation Falcon tubes (e.g., Fischer Scientific 14-432-22). Halocarbon oil 700 for injections (Sigma, cat. no. H8898). Optional reagents for validating insertions Potassium chloride (KCl, standard lab quality). Tris-HCl (pH 8.3, standard lab quality). Magnesium-chloride (MgCl 2 , standard lab quality). Nonidet P-40 (NP40, standard lab quality). Tween 20 (standard lab quality). Gelatin (standard lab quality). Proteinase K 10 mg/mL(Fisher Scientific, cat. no. EO0491) DNeasy blood and tissue kit (Qiagen, cat. no 69504). PCR amplification primers (see Table 4 ). dNTPs (NEB cat. no. N0447S) Phusion DNA polymerase (NEB cat. no. M0530S) Equipment Standard C. elegans injection setup: inverted microscope, injector, micromanipulator, injection needles, and 2% agarose slides for mounting animals. Standard dissection microscope for manipulating and visualizing C. elegans . C. elegans "worm pick" with platinum wire and flame for sterilizing. Fluorescence dissection microscope (GFP and RFP filter set). Optional: shutter controlled by a foot peddle. 20°C, 25°C, and 30°C incubators. Standard molecular biology equipment ( e.g. , pipettors, microcentrifuge, 1.5 mL microcentrifuge tubes, gel electrophoresis) Thermocycler for PCR. Reagent setup Hygromycin B solution Make 500 mL 4 mg/ul hygromycin stock solution. Dilute hygromycin in milliQ or distilled water. Use a 500 mL sterile vacuum filter to sterilize the solution. Aliquot into 50 mL Falcon tubes and store at -20°C until use. Worm lysis buffer (WLB) Make 1 mL of WLB with the following concentrations: 50mM KCl, 10mM Tris-HCl (pH8.3), 2.5mM MgCl2, 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% (w/v) Gelatin. Store as 100 ul aliquots. Procedure Generate MosTI target vectors (~ a few days - a month for gene synthesis) 1. Clone your transgene of interest into the appropriate target vector ( table 1 ). 2. Validate transgene inserted in target vector by standard methods (e.g., restriction digest, electrophoresis, Sanger sequencing) 3. Isolate plasmid DNA for injection using PureLink HQ mini plasmid purification kit. One 25 ul injection mix will require <100 ng plasmid. Prepare MosTI injection strains (~ one week before injection) 4. Maintain the appropriate MosTI injection strains ( table 2 ) in a healthy and fed state on HB101 or OP50 bacteria at 20°C. unc-119 animals are healthier on HB101 plates, especially if you pick a few animals to a new plate daily. Prepare MosTI injection mixes (the day of injection) 5. Make an injection mix for the appropriate selection strategy. 6. Spin all DNA preps at the highest speed on a tabletop centrifuge before making the injection mix. Spin injection mix again immediately before injection to minimize clogging injection needles. Day 1: MosTI injections (1-2 hours) 7. Inject young adult hermaphrodites with the appropriate strain ( i.e. , match the target vector with the split selection you are using, see Table 2 ) using standard injection techniques. 8. Place injected animals at room temperature to recover for approximately one hour. 9. After the recovery period, pick injected animal to HB101 or OP50 plates and place it in a 25°C incubator. You can place one or more injected animals on each plate (three injected worms on each plate work well in our hands). Day 3: Antibiotic selection (1 hour) 10. Forty-eight hours after injection, add 500 ul of 4 mg/ml hygromycin to the P0 plates to select transgenic array animals. Place plates in a clean area, leaving the lids off until the liquid has dried. Close the plates and place them in a 25°C incubator. ~Day 6-7: Heat shock transgenic animals (18 hours) 11. When the bacterial food is close to being exhausted, place all NGM injection plates in a 30°C air incubator for 18 hours to induce Cas9 expression. Note: we frequently observe plates with MosTI insertions, even in the absence of heat shock. We assume this is due to leaky expression from the heat-shock promoter or, possibly, starvation stress for unc-119 animals that cannot move. Suppose you need to make more independent MosTI insertions. In that case, you can pick animals with no single-copy insertion ( i.e. , unc-119 animals or non-fluorescent P mlc-2 :: gfp animals) and grow them for another generation before the heat shock. ~Day 7- 10. Screen for MosTI insertions (1 hour) 12. The screen for insertions is based on either Unc rescue (split unc-119 ) or GFP expression in all muscles (split Pmlc-2::gfp ). Screen for MosTI insertions when the food is exhausted on the NGM plates. Unc rescue is easiest screened on a standard dissection microscopy, whereas screening for GFP expression requires a fluorescence dissection microscope. 13. Pick a couple of putative insertion animals off each plate to a new plate. We pick more than one animal because we find a fraction of sterility in the injected worms. Since there may be independent inserts on a single plate (especially if you placed several injected animals on each plate) be sure to pick a single, clonal animal at a later generation to ensure that you are working with only one independent insertion. If possible, check the strain for the presence of your transgene (by fluorescence, for example). 14. Generate homozygous transgenic animals. You can do this by picking single animals to individual plates (~6) and screening for homozygous animals. Alternatively, you can propagate the strain for a couple of generations by "chunking," and the animals will tend to become homozygous on their own. Select only one homozygous plate as an independent insert from each injected worm. Recommended: validate full-length MosTI insertion by Sanger sequencing (2 days) The repair process generates a fraction of truncated or duplicated transgene insertions (approx. 15% total), and we recommend validating insertions. 15. Make crude genomic DNA lysate. Mix: 10 adult worms + 1 ul of Proteinase K (20 mg/mL) + 9 ul of WLB in a PCR tube. Freeze crack the worms by placing a PCR tube at -80°C for 10 minutes. Lyse the worms in a thermocycler with the following program: 60 min at 65°C and 15 min at 95°C. 16. PCR amplify transgene insertion. Amplify the transgene with the flanking sequence. See Table 4 for PCR primers corresponding to every MosTI insertion site. Note: For long transgenes, higher-quality DNA may facilitate PCR amplification. We use the Qiagen Dneasy blood and tissue kit when necessary. 17. Sanger sequencing. Verify the expected size of the transgene insert and validate the sequencing by Sanger sequencing using transgene-specific primers. Remove selection marker (1 week) The selection markers are flanked by LoxP sites and can be removed by injection of plasmids encoding Cre recombinase. In some cases, removing the selection marker may be beneficial ( e.g. , for a second round of transgenesis using the same selection marker or to remove fluorescence from split P mlc-2 :: gfp ). 18. Make Cre recombinase injection mix. 19. Inject homozygous MosTI transgenic animals and place single P0 injected animals on individual plates. 20. Optional. Two days later, add 500 ul of stock hygromycin selection to plates to select for Cre recombinase arrays. The Cre recombination efficiency is high enough that this step is not necessary but will enrich for animals with the split-selection marker removed. 21. Approx. five days later, screen F2 animals for removal of the selection marker (i.e., Unc animals for split unc-119 selection or non-fluorescent animals for P mlc-2 :: gfp selection). Recommended: outcross transgenic strains (1 week) 22. The insertion strains are relatively "clean" and were whole genome sequenced (El Mouridi et al., 2022). You can inspect the injection strains for SNPs that may affect your study's biological process. However, outcrossing is always a good habit. Anticipated Results Number of MosTI insertions From 10 transgenic lines, we generated five independent insertions (50% insertion rate, N = 2). Note that we did not optimize for insertion frequency. You could, in principle, establish just a few independent lines, split those into many different NGM plates, and perform the heat shock to generate more independent MosTI lines. The insertion frequency appears to depend on the length of the transgene. We have routinely generated 10 kb insertions, but others have reported trouble inserting larger transgenes. If you need to insert large single-copy transgenes, consider generating random insertions using the miniMos transposon, which can carry more than 40 kb transgenes (Frøkjær-Jensen et al., 2014). Full-length insertions In our hands, most single-copy insertions are full-length (approx. 80%). Still, validating the integrity and copy number of MosTI insertions is good practice if your experiments rely on just a single copy ( e.g. , comparing expression levels between different transgenes). In many cases, generating more than one MosTI insertion and ensuring all inserts behave similarly is more time-efficient (similar to having more than one mutant allele when studying a gene). Removing selection marker Expressing the PATC-rich Cre recombinase from arrays is efficient, and the excision process happens at a high frequency. We observe animals without the selection marker on most NGM plates with stable extra-chromosomal arrays containing the Cre plasmid. Declarations Acknowledgments We thank members of the SGB lab for MosTI testing and feedback. We thank Mohammed Aljohani for unpublished reagents (pMDJ39 and pMDJ231) and Ramatoulaye Balde for excellent lab support. Research in CFJ's laboratory is supported by KAUST core funding and KAUST's Office of Sponsored Research (OSR-CRG2020-4388). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440) and WormBase for data and literature curation. Author contributions SEM and CFJ developed the MosTI methods and protocols described. SEM wrote the first draft of the protocol and CFJ edited the final draft. SEM and CFJ generated figures. ORCID iD Sonia El Mouridi: https://orcid.org/0000-0002-3674-3151 Christian Frøkjær-Jensen: https://orcid.org/0000-0002-3178-0906 Competing interests The authors declare no competing interests. References Aljohani, M.D., El Mouridi, S., Priyadarshini, M., Vargas-Velazquez, A.M., and Frøkjær-Jensen, C. (2020). Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in C. elegans . Nat Commun 11 , 6300. https://doi.org/10.1038/s41467-020-19898-0. 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Semple, J.I., Biondini, L., and Lehner, B. (2012). Generating transgenic nematodes by bombardment and antibiotic selection. Nat. Methods 9 , 118–119. https://doi.org/10.1038/nmeth.1864. Stevenson, Z.C., Moerdyk-Schauwecker, M.J., Jamison, B., and Phillips, P.C. (2020). Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans . G3 Genes|Genomes|Genetics 10 , 3775–3782. https://doi.org/10.1534/g3.120.401400. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 1–11. https://doi.org/10.1895/wormbook.1.101.1. Stinchcomb, D.T., Shaw, J.E., Carr, S.H., and Hirsh, D. (1985). Extrachromosomal DNA transformation of Caenorhabditis elegans . Mol. Cell. Biol. 5 , 3484–3496. . Stringham, E.G., Dixon, D.K., Jones, D., and Candido, E.P. (1992). Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans. MBoC 3 , 221–233. https://doi.org/10.1091/mbc.3.2.221. Tyson, J.R., O’Neil, N.J., Jain, M., Olsen, H.E., Hieter, P., and Snutch, T.P. (2018). MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome. Genome Res. 28 , 266–274. https://doi.org/10.1101/gr.221184.117. Vallin, E., Gallagher, J., Granger, L., Martin, E., Belougne, J., Maurizio, J., Duverger, Y., Scaglione, S., Borrel, C., Cortier, E., et al. (2012). A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community. PLoS ONE 7 , e30482. https://doi.org/10.1371/journal.pone.0030482. Zeiser, E., Frøkjær-Jensen, C., Jorgensen, E., and Ahringer, J. (2011). MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline. PLoS ONE 6 , e20082. https://doi.org/10.1371/journal.pone.0020082. Tables Tables 1-4 are available in the supplementary files. Additional Images Additional images are available in the supplementary files. 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The simple injection of DNA fragments (linear or circular) generates heritable extra-chromosomal arrays containing many DNA copies at high frequency\u0026nbsp;(Stinchcomb et al., 1985). Optimized injection conditions (\u003cem\u003ee.g.\u003c/em\u003e, DNA concentration and the identification of a genetic selection marker)(Mello et al., 1991)\u0026nbsp;have made transgenesis routine in many \u003cem\u003eC. elegans\u003c/em\u003e laboratories. Extra-chromosomal arrays have some clear advantages: somatic transgene expression is frequently high due to transgene copy number (approx. 100 plasmids in an array), \u0026nbsp;the genome is unaltered, and arrays are easy to lose with no requirement for genetic crosses due to mitotic loss in cell divisions. The disadvantages of arrays include the requirement for continuous selection, highly variable expression, and transgenes that are often silenced in the germline\u0026nbsp;(Kelly et al., 1997). It is possible to partially mitigate germline array silencing by adding complex DNA to the injection mix\u0026nbsp;(Kelly et al., 1997)\u0026nbsp;or by linearizing plasmids and including a class of non-coding DNA structures (PATCs)\u0026nbsp;(Aljohani et al., 2020; Fire et al., 2006; Fr\u0026oslash;kj\u0026aelig;r-Jensen, 2021; Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2016), but these manipulations are somewhat cumbersome.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Genomic insertion of low or single-copy transgenes can overcome many of the limitations of arrays. Random low-copy insertions can be generated by biolistic transformation using genetic selection markers\u0026nbsp;(Praitis et al., 2001)\u0026nbsp;or antibiotic selection\u0026nbsp;(Radman et al., 2013; Semple et al., 2012). Some transgenic animals generated by biolistic transformation show stable germline expression\u0026nbsp;(Merritt et al., 2008; Praitis et al., 2001), but transgene insertions are inherently mutagenic with occasional large-scale chromosome duplications, and transgenes are often truncated\u0026nbsp;(Tyson et al., 2018). Transgenes can also be inserted at random locations by transposition with an optimized Mos1 transposon\u0026nbsp;(Fr\u0026oslash;kj\u0026aelig;r-Jensen, 2015; Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2014), which has the advantage that the exact insertion sites are easily determined\u0026nbsp;(Boulin and Bessereau, 2007). However, the random nature of the transgene insertion site results in position effect variegation evidenced by variable somatic expression and frequent germline silencing(Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2016).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;For these reasons, we and others have developed targeted-single copy transgene insertion methods based on homologous repair at double-strand DNA breaks (DSBs). The initial methods for modifying the genome were based on generating DSBs at a well-defined location using a native\u0026nbsp;(Barrett et al., 2004)\u0026nbsp;or exogenous Mos1 transposons\u0026nbsp;(Bessereau et al., 2001; Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2008, 2010; Robert and Bessereau, 2007).\u0026nbsp;Using an extensive collection of strains with mapped Mos1 insertions\u0026nbsp;(Vallin et al., 2012), it was possible to identify safe-harbor insertion sites, where transgenes were expressed at native levels and in the germline from the \u003cem\u003epie-1\u0026nbsp;\u003c/em\u003e(Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2008)\u0026nbsp;or \u003cem\u003emex-5\u0026nbsp;\u003c/em\u003e(Zeiser et al., 2011)\u0026nbsp;promoters.\u0026nbsp;Mos1-mediated single-copy transgene insertions (MosSCI) could be generated at a reasonably high frequency using genetic\u0026nbsp;(Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2008, 2012)\u0026nbsp;and antibiotic\u0026nbsp;(Giordano-Santini et al., 2010; Semple et al., 2010)\u0026nbsp;selection markers.\u0026nbsp;Later MosSCI iterations used similar selection strategies but generated DSBs using CRISPR/Cas9\u0026nbsp;(Dickinson and Goldstein, 2016; Dickinson et al., 2013; Fr\u0026oslash;kj\u0026aelig;r-Jensen, 2013). More recently, methods to insert single-copy transgenes based on recombination-mediated cassette exchange have also been developed\u0026nbsp;(Nonet, 2021, 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;One bottleneck for these targeted insertion methods is to distinguish between positive selection from arrays and single-copy inserts (\u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003eunc-119\u003c/em\u003e rescue could be conferred by the intermediate extra-chromosomal arrays used to generate single-copy insertions). Selecting single-copy insertions is facilitated by co-injecting a single muscle-expressed fluorescent marker\u0026nbsp;(El Mouridi et al., 2020)\u0026nbsp;or co-injection negative array selection markers, such as inducible expression of a toxin\u0026nbsp;(Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2012)\u0026nbsp;or drug-inducible paralysis\u0026nbsp;(El Mouridi et al., 2021). However, both approaches require significant screening or additional manipulations, respectively. To facilitate the identification of single-copy insertions, we and others have recently developed methods that rely on \u0026quot;split\u0026quot; selection markers\u0026nbsp;(El Mouridi et al., 2022; Stevenson et al., 2020). With these methods, selection markers are split between the transgene vector and the safe-harbor landing site: the selection marker is only reconstituted and functional when a transgene is correctly insertion, which significantly facilitates screening for insertions (\u003cstrong\u003eFigure 1\u003c/strong\u003e). The split selection markers include antibiotic (hygromycin)\u0026nbsp;(Stevenson et al., 2020), genetic (\u003cem\u003eunc-119\u003c/em\u003e), and visual markers (GFP fluorescence)\u0026nbsp;(El Mouridi et al., 2022; Stevenson et al., 2020). Both split-selection methods rely on CRISPR/Cas9 expression to induce DSBs, but the methods differ in several respects that favor different experimental setups. Stevenson \u003cem\u003eet al\u003c/em\u003e.(2020)\u0026nbsp;developed a split antibiotic selection marker (hygromycin) and focused on generating transgene insertions based on \u003cem\u003ein situ\u003c/em\u003e assembly from several PCR fragments. Their method has the advantage of injecting into wild-type animals and an assembly method that requires no cloning, which, in principle, can be multiplexed. The disadvantages are that insertion frequencies are relatively low, homozygous animals are not as easy to generate using antibiotic selection, and the \u003cem\u003ein situ\u003c/em\u003e assemblies are error-prone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;We developed MosTI with the intention of incorporating several incremental single-copy transgenesis improvements, including split-selection markers, developed across many laboratories. We aimed to generate a set of standardized reagents, with several validated safe-harbor landing sites permissive for germline expression, an increased number of split selection markers (\u003cem\u003eunc-119\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Pmlc-2::gfp\u003c/em\u003e), and a collection of target plasmids that contain common regulatory elements. Importantly, MosTI is compatible with single-copy transgene insertion and targeted array integration. Furthermore, the safe-harbor landing sites are modular and can be converted from one selection to another for added flexibility. One key feature of MosTI is the ability to generate DSBs with Cas9 using several strategies: constitutive expression of a PATC-rich Cas9, heat-shock inducible Cas9 expression from linearized transgenes, and two-step injections of Cas9 protein complexed with crRNA/tracrRNAs.\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Two pairs of divergently transcribed heat-shock promoters\u0026nbsp;(Russnak and Candido, 1985), \u003cem\u003ehsp-16.48\u003c/em\u003e/\u003cem\u003ehsp-16.1\u003c/em\u003e and \u003cem\u003ehsp-16.41\u003c/em\u003e/\u003cem\u003ehsp-16.2\u003c/em\u003e, are commonly used for heat-inducible expression of transgenes in \u003cem\u003eC. elegans\u0026nbsp;\u003c/em\u003e(Stringham et al., 1992). Extra-chromosomal arrays carrying transgenes expressed under any one of the \u003cem\u003ehsp\u003c/em\u003e promoters can drive inducible expression in most somatic cells at high levels after a short heat shock (1-2 hours at 33\u0026deg;C), but no expression was observed in the germ line or early embryo. Similarly, GFP expression from a single-copy \u003cem\u003ehsp\u003c/em\u003e transgene is bright in somatic cells but very dim in germ cells (Zeiser et al., 2011). The mechanisms that limit heat-shock expression in germ cells are not well-defined, but modified transgenes with PATCs (Aljohani et al., 2020) and an improved extended heat-shock protocol (Nonet, 2021) can improve inducible Cas9 expression (El Mouridi et al., 2022). Here, we describe a detailed protocol for generating single-copy transgene insertions using heat-shock inducible germline expression of Cas9 from arrays and selecting insertion events by propagating animals over generations.\u003c/p\u003e"},{"header":"Experimental Design","content":"\u003cp\u003eBrief overview\u003c/p\u003e\n\u003cp\u003eInserting a single-copy transgene by MosTI consists of several steps (\u003cstrong\u003eFigure 1\u003c/strong\u003e):\u003c/p\u003e\n\u003cp\u003e(1) \u003cstrong\u003eTransgene cloning\u003c/strong\u003e. Cloning transgenes into the appropriate target vector. The target vectors are specific to the split selection marker (\u003cem\u003ei.e.\u003c/em\u003e, there are different vectors for \u003cem\u003eunc-119\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Pmlc-2::gfp\u003c/em\u003e). However, using a given selection marker, the target vectors are universal for all safe-harbor landing sites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(2) \u003cstrong\u003eMicro-injections and identifying MosTI insertions\u003c/strong\u003e. This step requires injecting plasmids into the appropriate MosTI strain using standard techniques, selecting for arrays, and screening for single-copy transgene insertion.\u003c/p\u003e\n\u003cp\u003e(3) \u003cstrong\u003eInsert validation (optional but recommended)\u003c/strong\u003e. The final steps include removal of the selection marker, validating the integrity of the transgene insertion, and genetic outcrossing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNecessary expertise\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eFundamental molecular biology expertise is required to clone transgenes into targeting vectors, \u003cem\u003ee.g.\u003c/em\u003e, restriction enzyme cloning, Golden-gate cloning\u0026nbsp;(Engler et al., 2008), or Gibson assembly\u0026nbsp;(Gibson et al., 2009). Alternatively, transgenes can be generated directly by gene synthesis. We have deposited a MosTI target vector for split \u003cem\u003eunc-119\u003c/em\u003e selection (pSEM246) at Twist Biosciences, which is available for custom vector gene synthesis by email request to the corresponding author.\u003c/li\u003e\n \u003cli\u003eBasic expertise in \u003cem\u003eC. elegans\u003c/em\u003e microinjection is required. The protocol was optimized to minimize the number of injections required by allowing inducible germline Cas9 expression from arrays; thus, only a few transgenic lines are necessary for generating several independent MosTI insertions.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eLimitations\u003c/h3\u003e\n\u003cp\u003eTransgenes can serve essential experimental functions but may only approximate how endogenous genes function. For example, regulatory elements may be position-dependent, and the usage of selection markers could have subtle effects on animals that are difficult to identify. Also, \u003cem\u003eC. elegans\u003c/em\u003e have many competing small RNA and non-coding DNA pathways that enable germ cells to distinguish between endogenous and foreign DNA, such as transgenes (Fr\u0026oslash;kj\u0026aelig;r-Jensen, 2019). We suggest that experimenters use common sense and critically evaluate their results, preferably by having separate lines of experiments that do not depend on transgenes to support or refute observations. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Materials","content":"\u003ch3\u003eBiological materials\u003c/h3\u003e\n\u003cp\u003eRequired plasmids\u003c/p\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eMosTI target vectors: pSEM246 (\u003cem\u003eunc-119\u003c/em\u003e selection) or pSEM272 (P\u003cem\u003emlc-2\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e selection)\u003c/li\u003e\n \u003cli\u003eCo-injection plasmids: pSEM231 (P\u003cem\u003emlc-1\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e), pSEM233 (P\u003cem\u003emlc-1\u003c/em\u003e::\u003cem\u003etagRFP\u003c/em\u003e), and pCFJ782 (HygroR).\u003c/li\u003e\n \u003cli\u003eCas9 plasmid: pMDJ231 (P\u003cem\u003ehsp-16.41\u003c/em\u003e::\u003cem\u003ecas9::gpd-2::tagRFP\u003c/em\u003e).\u003c/li\u003e\n \u003cli\u003esgRNA plasmids: pSEM318 (split \u003cem\u003eunc-119\u003c/em\u003e) or pSEM255 (split P\u003cem\u003emlc-2\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e)\u003c/li\u003e\n \u003cli\u003e1 kb Plus DNA ladder (500 ng/ul, no dye, Invitrogen #10787018)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNote: see \u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003efor all MosTI plasmids and their Addgene IDs. Some target vectors with common promoters and fluorophores may facilitate generating transgenes following common design rules (Nance and Fr\u0026oslash;kj\u0026aelig;r-Jensen, 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eMosTI injection strains (see \u003cstrong\u003eTable 2\u003c/strong\u003e). Strains are distributed by the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e Genetics Center (CGC).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOP50 and HB101 bacterial cultures. These are distributed by the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e Genetics Center (CGC).\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cul\u003e\n \u003cli\u003eMilli-Q water or distilled water.\u003c/li\u003e\n \u003cli\u003eNematode Growth Medium (NGM). Standard recipe as described in Stiernagle (2006).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e6 cm vented Petri dishes for NGM plates.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStandard molecular biology reagents (\u003cem\u003ee.g.\u003c/em\u003e, restriction enzymes to generate and validate transgenes).\u003c/li\u003e\n \u003cli\u003eApaLI (NEB cat. no. R0507S).\u003c/li\u003e\n \u003cli\u003ePureLink HQ mini plasmid purification kit (cat. no. K2100-01).\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNote: Higher purity DNA can improve single-copy insertion frequencies (Fr\u0026oslash;kj\u0026aelig;r-Jensen, unpub. obs.), but you can use any commercial miniprep kit.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eMonarch PCR \u0026amp; DNA Cleanup Kit (NEB cat. no. T1030S).\u003c/li\u003e\n \u003cli\u003eHygromycin B (Gold Biotechnology, cat. no. H270-1).\u003c/li\u003e\n \u003cli\u003e500 mL single-use sterile vacuum filter (\u003cem\u003ee.g.\u003c/em\u003e, Fisher Scientific 09-741-20200).\u003c/li\u003e\n \u003cli\u003e50 mL conical centrifugation Falcon tubes (e.g., Fischer Scientific 14-432-22).\u003c/li\u003e\n \u003cli\u003eHalocarbon oil 700 for injections (Sigma, cat. no. H8898).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eOptional reagents for validating insertions\u003c/h3\u003e\n\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003ePotassium chloride (KCl, standard lab quality).\u003c/li\u003e\n \u003cli\u003eTris-HCl (pH 8.3, standard lab quality).\u003c/li\u003e\n \u003cli\u003eMagnesium-chloride (MgCl\u003csub\u003e2\u003c/sub\u003e, standard lab quality).\u003c/li\u003e\n \u003cli\u003eNonidet P-40 (NP40, standard lab quality).\u003c/li\u003e\n \u003cli\u003eTween 20 (standard lab quality).\u003c/li\u003e\n \u003cli\u003eGelatin (standard lab quality).\u003c/li\u003e\n \u003cli\u003eProteinase K 10 mg/mL(Fisher Scientific, cat. no. EO0491)\u003c/li\u003e\n \u003cli\u003eDNeasy blood and tissue kit (Qiagen, cat. no 69504).\u003c/li\u003e\n \u003cli\u003ePCR amplification primers (see \u003cstrong\u003eTable 4\u003c/strong\u003e).\u003c/li\u003e\n \u003cli\u003edNTPs (NEB cat. no. N0447S)\u003c/li\u003e\n \u003cli\u003ePhusion DNA polymerase (NEB cat. no. M0530S)\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eEquipment\u003c/h3\u003e\n\u003cul\u003e\n \u003cli\u003eStandard \u003cem\u003eC. elegans\u003c/em\u003e injection setup: inverted microscope, injector, micromanipulator, injection needles, and 2% agarose slides for mounting animals.\u003c/li\u003e\n \u003cli\u003eStandard dissection microscope for manipulating and visualizing \u003cem\u003eC. elegans\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eC. elegans\u003c/em\u003e \u0026quot;worm pick\u0026quot; with platinum wire and flame for sterilizing.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFluorescence dissection microscope (GFP and RFP filter set). Optional: shutter controlled by a foot peddle.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e20\u0026deg;C, 25\u0026deg;C, and 30\u0026deg;C incubators.\u003c/li\u003e\n \u003cli\u003eStandard molecular biology equipment (\u003cem\u003ee.g.\u003c/em\u003e, pipettors, microcentrifuge, 1.5 mL microcentrifuge tubes, gel electrophoresis)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThermocycler for PCR.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eReagent setup\u003c/h3\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eHygromycin B solution\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eMake 500 mL 4 mg/ul hygromycin stock solution. Dilute hygromycin in milliQ or distilled water. Use a 500 mL sterile vacuum filter to sterilize the solution. Aliquot into 50 mL Falcon tubes and store at -20\u0026deg;C until use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eWorm lysis buffer (WLB)\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eMake 1 mL of WLB with the following concentrations: 50mM KCl, 10mM Tris-HCl (pH8.3), 2.5mM MgCl2, 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% (w/v) Gelatin. Store as 100 ul aliquots.\u003c/p\u003e"},{"header":"Procedure","content":"\u003cp\u003e\u003cstrong\u003eGenerate MosTI target vectors\u0026nbsp;\u003c/strong\u003e(~ a few days - a month for gene synthesis)\u003c/p\u003e\n\u003cp\u003e1. Clone your transgene of interest into the appropriate target vector (\u003cstrong\u003etable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e2. Validate transgene inserted in target vector by standard methods (e.g., restriction digest, electrophoresis, Sanger sequencing)\u003c/p\u003e\n\u003cp\u003e3. Isolate plasmid DNA for injection using PureLink HQ mini plasmid purification kit. One 25 ul injection mix will require \u0026lt;100 ng plasmid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePrepare MosTI injection strains\u0026nbsp;\u003c/strong\u003e(~ one week before injection)\u003c/p\u003e\n\u003cp\u003e4. Maintain the appropriate MosTI injection strains (\u003cstrong\u003etable 2\u003c/strong\u003e) in a healthy and fed state on HB101 or OP50 bacteria at 20\u0026deg;C. \u003cem\u003eunc-119\u003c/em\u003e animals are healthier on HB101 plates, especially if you pick a few animals to a new plate daily.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrepare MosTI injection mixes\u0026nbsp;\u003c/strong\u003e(the day of injection)\u003c/p\u003e\n\u003cp\u003e5. Make an injection mix for the appropriate selection strategy.\u003c/p\u003e\n\u003cp\u003e6. Spin all DNA preps at the highest speed on a tabletop centrifuge before making the injection mix. Spin injection mix again immediately before injection to minimize clogging injection needles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDay 1: MosTI injections\u0026nbsp;\u003c/strong\u003e(1-2 hours)\u003c/p\u003e\n\u003cp\u003e7. Inject young adult hermaphrodites with the appropriate strain (\u003cem\u003ei.e.\u003c/em\u003e, match the target vector with the split selection you are using, see \u003cstrong\u003eTable 2\u003c/strong\u003e) using standard injection techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e8. Place injected animals at room temperature to recover for approximately one hour.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e9. After the recovery period, pick injected animal to HB101 or OP50 plates and place it in a 25\u0026deg;C incubator. You can place one or more injected animals on each plate (three injected worms on each plate work well in our hands).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDay 3: Antibiotic selection\u0026nbsp;\u003c/strong\u003e(1 hour)\u003c/p\u003e\n\u003cp\u003e10. Forty-eight hours after injection, add 500 ul of 4 mg/ml hygromycin to the P0 plates to select transgenic array animals. Place plates in a clean area, leaving the lids off until the liquid has dried. Close the plates and place them in a 25\u0026deg;C incubator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e~Day 6-7: Heat shock transgenic animals\u0026nbsp;\u003c/strong\u003e(18 hours)\u003c/p\u003e\n\u003cp\u003e11. When the bacterial food is close to being exhausted, place all NGM injection plates in a 30\u0026deg;C air incubator for 18 hours to induce Cas9 expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: we frequently observe plates with MosTI insertions, even in the absence of heat shock. We assume this is due to leaky expression from the heat-shock promoter or, possibly, starvation stress for \u003cem\u003eunc-119\u003c/em\u003e animals that cannot move. Suppose you need to make more independent MosTI insertions. In that case, you can pick animals with no single-copy insertion (\u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003eunc-119\u003c/em\u003e animals or non-fluorescent P\u003cem\u003emlc-2\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e animals) and grow them for another generation before the heat shock.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e~Day 7- 10. Screen for MosTI insertions\u0026nbsp;\u003c/strong\u003e(1 hour)\u003c/p\u003e\n\u003cp\u003e12. The screen for insertions is based on either Unc rescue (split \u003cem\u003eunc-119\u003c/em\u003e) or GFP expression in all muscles (split\u003cem\u003e\u0026nbsp;Pmlc-2::gfp\u003c/em\u003e). Screen for MosTI insertions when the food is exhausted on the NGM plates. Unc rescue is easiest screened on a standard dissection microscopy, whereas screening for GFP expression requires a fluorescence dissection microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e13. Pick a couple of putative insertion animals off each plate to a new plate. We pick more than one animal because we find a fraction of sterility in the injected worms. Since there may be independent inserts on a single plate (especially if you placed several injected animals on each plate) be sure to pick a single, clonal animal at a later generation to ensure that you are working with only one independent insertion. If possible, check the strain for the presence of your transgene (by fluorescence, for example).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e14. Generate homozygous transgenic animals. You can do this by picking single animals to individual plates (~6) and screening for homozygous animals. Alternatively, you can propagate the strain for a couple of generations by \u0026quot;chunking,\u0026quot; and the animals will tend to become homozygous on their own. Select only one homozygous plate as an independent insert from each injected worm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecommended: validate full-length MosTI insertion by Sanger sequencing\u0026nbsp;\u003c/strong\u003e(2 days)\u003c/p\u003e\n\u003cp\u003eThe repair process generates a fraction of truncated or duplicated transgene insertions (approx. 15% total), and we recommend validating insertions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e15. Make crude genomic DNA lysate.\u003c/p\u003e\n\u003cp\u003eMix: 10 adult worms + 1 ul of Proteinase K (20 mg/mL) + 9 ul of WLB in a PCR tube.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFreeze crack the worms by placing a PCR tube at -80\u0026deg;C for 10 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLyse the worms in a thermocycler with the following program: 60 min at 65\u0026deg;C and 15 min at 95\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e16. PCR amplify transgene insertion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmplify the transgene with the flanking sequence. See \u003cstrong\u003eTable 4\u003c/strong\u003e for PCR primers corresponding to every MosTI insertion site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote: For long transgenes, higher-quality DNA may facilitate PCR amplification. We use the Qiagen Dneasy blood and tissue kit when necessary.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e17. Sanger sequencing.\u003c/p\u003e\n\u003cp\u003eVerify the expected size of the transgene insert and validate the sequencing by Sanger sequencing using transgene-specific primers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemove selection marker\u0026nbsp;\u003c/strong\u003e(1 week)\u003c/p\u003e\n\u003cp\u003eThe selection markers are flanked by LoxP sites and can be removed by injection of plasmids encoding Cre recombinase. In some cases, removing the selection marker may be beneficial (\u003cem\u003ee.g.\u003c/em\u003e, for a second round of transgenesis using the same selection marker or to remove fluorescence from split P\u003cem\u003emlc-2\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e18. Make Cre recombinase injection mix.\u003c/p\u003e\n\u003cp\u003e19. Inject homozygous MosTI transgenic animals and place single P0 injected animals on individual plates.\u003c/p\u003e\n\u003cp\u003e20. Optional. Two days later, add 500 ul of stock hygromycin selection to plates to select for Cre recombinase arrays. The Cre recombination efficiency is high enough that this step is not necessary but will enrich for animals with the split-selection marker removed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e21. Approx. five days later, screen F2 animals for removal of the selection marker (i.e., Unc animals for split \u003cem\u003eunc-119\u003c/em\u003e selection or non-fluorescent animals for P\u003cem\u003emlc-2\u003c/em\u003e::\u003cem\u003egfp\u003c/em\u003e selection).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecommended: outcross transgenic strains\u0026nbsp;\u003c/strong\u003e(1 week)\u003c/p\u003e\n\u003cp\u003e22. The insertion strains are relatively \u0026quot;clean\u0026quot; and were whole genome sequenced (El Mouridi et al., 2022). You can inspect the injection strains for SNPs that may affect your study\u0026apos;s biological process. However, outcrossing is always a good habit.\u003c/p\u003e"},{"header":"Anticipated Results","content":"\u003ch3\u003eNumber of MosTI insertions\u003c/h3\u003e\n\u003cp\u003eFrom 10 transgenic lines, we generated five independent insertions (50% insertion rate, N = 2). Note that we did not optimize for insertion frequency. You could, in principle, establish just a few independent lines, split those into many different NGM plates, and perform the heat shock to generate more independent MosTI lines. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The insertion frequency appears to depend on the length of the transgene. We have routinely generated 10 kb insertions, but others have reported trouble inserting larger transgenes. If you need to insert large single-copy transgenes, consider generating random insertions using the miniMos transposon, which can carry more than 40 kb transgenes\u0026nbsp;(Fr\u0026oslash;kj\u0026aelig;r-Jensen et al., 2014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eFull-length insertions\u003c/h3\u003e\n\u003cp\u003eIn our hands, most single-copy insertions are full-length (approx. 80%). Still, validating the integrity and copy number of MosTI insertions is good practice if your experiments rely on just a single copy (\u003cem\u003ee.g.\u003c/em\u003e, comparing expression levels between different transgenes). In many cases, generating more than one MosTI insertion and ensuring all inserts behave similarly is more time-efficient (similar to having more than one mutant allele when studying a gene).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eRemoving selection marker\u003c/h3\u003e\n\u003cp\u003eExpressing the PATC-rich Cre recombinase from arrays is efficient, and the excision process happens at a high frequency. We observe animals without the selection marker on most NGM plates with stable extra-chromosomal arrays containing the Cre plasmid.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank members of the SGB lab for MosTI testing and feedback. We thank Mohammed Aljohani for unpublished reagents (pMDJ39 and pMDJ231) and Ramatoulaye Balde for excellent lab support. Research in CFJ\u0026apos;s laboratory is supported by KAUST core funding and KAUST\u0026apos;s Office of Sponsored Research (OSR-CRG2020-4388). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440)\u0026nbsp;and WormBase for data and literature curation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eSEM and CFJ developed the MosTI methods and protocols described. SEM wrote the first draft of the protocol and CFJ edited the final draft. SEM and CFJ generated figures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eORCID iD\u003c/h2\u003e\n\u003cp\u003eSonia El Mouridi: https://orcid.org/0000-0002-3674-3151\u003c/p\u003e\n\u003cp\u003eChristian Fr\u0026oslash;kj\u0026aelig;r-Jensen: https://orcid.org/0000-0002-3178-0906\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAljohani, M.D., El Mouridi, S., Priyadarshini, M., Vargas-Velazquez, A.M., and Fr\u0026oslash;kj\u0026aelig;r-Jensen, C. (2020). Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in \u003cem\u003eC. elegans\u003c/em\u003e. Nat Commun \u003cem\u003e11\u003c/em\u003e, 6300. https://doi.org/10.1038/s41467-020-19898-0.\u003c/p\u003e\n\u003cp\u003eBarrett, P.L., Fleming, J.T., and G\u0026ouml;bel, V. (2004). Targeted gene alteration in Caenorhabditis elegans by gene conversion. Nat. Genet. \u003cem\u003e36\u003c/em\u003e, 1231\u0026ndash;1237. https://doi.org/10.1038/ng1459.\u003c/p\u003e\n\u003cp\u003eBessereau, J.-L., Wright, A., Williams, D.C., Schuske, K., Davis, M.W., and Jorgensen, E.M. (2001). Mobilization of a \u003cem\u003eDrosophila\u003c/em\u003e transposon in the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e germ line. Nature \u003cem\u003e413\u003c/em\u003e, 70\u0026ndash;74. https://doi.org/10.1038/35092567.\u003c/p\u003e\n\u003cp\u003eBoulin, T., and Bessereau, J.-L. (2007). Mos1-mediated insertional mutagenesis in Caenorhabditis elegans. Nat Protoc \u003cem\u003e2\u003c/em\u003e, 1276\u0026ndash;1287. https://doi.org/10.1038/nprot.2007.192.\u003c/p\u003e\n\u003cp\u003eDickinson, D.J., and Goldstein, B. (2016). CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering. Genetics \u003cem\u003e202\u003c/em\u003e, 885\u0026ndash;901. https://doi.org/10.1534/genetics.115.182162.\u003c/p\u003e\n\u003cp\u003eDickinson, D.J., Ward, J.D., Reiner, D.J., and Goldstein, B. (2013). Engineering the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e genome using Cas9-triggered homologous recombination.\u0026nbsp;Nat. Methods \u003cem\u003e10\u003c/em\u003e, 1028\u0026ndash;1034. https://doi.org/10.1038/nmeth.2641.\u003c/p\u003e\n\u003cp\u003eEl Mouridi, S., Peng, Y., and Fr\u0026oslash;kj\u0026aelig;r-Jensen, C. (2020).\u0026nbsp;Characterizing a strong pan-muscular promoter (P\u003cem\u003emlc-1\u003c/em\u003e) as a fluorescent co-injection marker to select for single-copy insertions. MicroPubl Biol \u003cem\u003e2020\u003c/em\u003e. https://doi.org/10.17912/micropub.biology.000302.\u003c/p\u003e\n\u003cp\u003eEl Mouridi, S., AlHarbi, S., and Fr\u0026oslash;kj\u0026aelig;r-Jensen, C. (2021). A histamine-gated channel is an efficient negative selection marker for \u003cem\u003eC. elegans\u003c/em\u003e transgenesis. MicroPubl Biol \u003cem\u003e2021\u003c/em\u003e. https://doi.org/10.17912/micropub.biology.000349.\u003c/p\u003e\n\u003cp\u003eEl Mouridi, S., Alkhaldi, F., and Fr\u0026oslash;kj\u0026aelig;r-Jensen, C. (2022). Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans. G3 (Bethesda) \u003cem\u003e12\u003c/em\u003e, jkac184. https://doi.org/10.1093/g3journal/jkac184.\u003c/p\u003e\n\u003cp\u003eEngler, C., Kandzia, R., and Marillonnet, S. (2008). A one pot, one step, precision cloning method with high throughput capability. 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PLoS ONE \u003cem\u003e6\u003c/em\u003e, e20082. https://doi.org/10.1371/journal.pone.0020082.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-4 are available in the supplementary files.\u003c/p\u003e"},{"header":"Additional Images","content":"\u003cp\u003eAdditional images are available in the supplementary files.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"6c7ddb5d-2bf9-4bff-90d1-569255ed2c9b","identifier":"10.13039/501100004052","name":"King Abdullah University of Science and Technology","awardNumber":"OSR-CRG2020-4388","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"King Abdullah University of Science and Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"C. elegans, genetic engineering, single-copy transgene, Cas9","lastPublishedDoi":"10.21203/rs.3.rs-2053151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2053151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrecise expression of transgenes in \u003cem\u003eC. elegans\u003c/em\u003e can be used to understand gene regulation, control cells, or as a starting point for genetic screens. Insertion of single-copy transgenes into well-defined safe-harbor locations is useful when consistent expression levels are required (\u003cem\u003ee.g.\u003c/em\u003e, to compare expression from different transgenes) or when expression is desired in germ cells. Here, we describe a detailed protocol for inserting single-copy transgenes using Modular Safe-harbor Transgene Insertion (MosTI) and a heat-shock inducible Cas9 expressed from a co-injected plasmid. Inducible Cas9 expression has the advantage of requiring few injections, and many independent single-copy insertions can be generated from a single array line.\u003c/p\u003e","manuscriptTitle":"MosTI single-copy transgene insertion in C. elegans using heat-shock inducible Cas9 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-14 19:26:10","doi":"10.21203/rs.3.rs-2053151/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30d00323-ad66-4889-a4ab-e712902cb98f","owner":[],"postedDate":"September 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":15434111,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2022-09-14T19:26:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-14 19:26:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2053151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2053151","identity":"rs-2053151","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}