An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9

Abstract

Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.

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References 44

1. 1.

   Arribere J. A. Bell R. T. Fu B. X. H. Artiles K. L. Hartman P. S., 2014 Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198: 837–846.

   Not in our library10.1534/genetics.114.169730

2. 2.

   Au V. Li-Leger E. Raymant G. Flibotte S. Chen G., 2018 Optimizing guide RNA selection and CRISPR/Cas9 methodology for efficient generation of deletions in C. elegans. bioRxiv.

   Not in our library10.1101/359588

3. 3.

   Brenner S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71–94.

   Not in our library10.1093/genetics/77.1.71

4. 4.
   Large-Scale Screening for Targeted Knockouts in the Caenorhabditis elegans Genome · 2012

   No notice
5. 5.
   Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination · 2013

   No notice
6. 6.

   Chen X. Xu F. Zhu C. Ji J. Zhou X., 2014 Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans. Sci. Rep. 4: 1–7.

   Not in our library10.1038/srep07581

7. 7.

   Chiu H. Schwartz H. T. Antoshechkin I. Sternberg P. W., 2013 Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics 195: 1167–1171.

   Not in our library10.1534/genetics.113.155879

8. 8.

   Cho S. W. Lee J. Carroll D. Kim J. S. Lee J., 2013 Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195: 1177–1180.

   Not in our library10.1534/genetics.113.155853

9. 9.

   Cong L. Ran F. A. Cox D. Lin S. Barretto R., 2013 Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339: 819–823.

   Not in our library10.1126/science.1231143

10. 10.
    A Transparent Window into Biology: A Primer on Caenorhabditis elegans · 2015

    No notice
11. 11.
    An Aneuploidy-Free and Structurally Defined Balancer Chromosome Toolkit for Caenorhabditis elegans · 2018

    Not checked
12. 12.

    Dickinson D. J. Ward J. D. Reiner D. J. Goldstein B., 2013 Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10: 1028–1034.

    Not in our library10.1038/nmeth.2641

13. 13.

    Dickinson D. J. Pani A. M. Heppert J. K. Higgins C. D. Goldstein B., 2015 Streamlined genome engineering with a self-excising drug selection cassette. Genetics 200: 1035–1049.

    Not in our library10.1534/genetics.115.178335

14. 14.

    Dickinson D. J. Goldstein B., 2016 CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics 202: 885–901.

    Not in our library10.1534/genetics.115.182162

15. 15.

    Farboud B. Meyer B. J., 2015 Dramatic enhancement of genome editing by CRISPR/cas9 through improved guide RNA design. Genetics 199: 959–971.

    Not in our library10.1534/genetics.115.175166

16. 16.

    Farboud B. Jarvis E. Roth T. L. Shin J. Corn J. E., 2018 Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms. J. Vis. Exp. (135).

    Not in our library10.3791/57350

17. 17.

    Friedland A. E. Tzur Y. B. Esvelt K. M. Colaiácovo M. P. Church G. M., 2013 Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10: 741–743.

    Not in our library10.1038/nmeth.2532

18. 18.

    Fu B. X. H. Hansen L. L. Artiles K. L. Nonet M. L. Fire A. Z., 2014 Landscape of target:guide homology effects on Cas9-mediated cleavage. Nucleic Acids Res. 42: 13778–13787.

    Not in our library10.1093/nar/gku1102

19. 19.
    Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs · 2014

    Corrected
20. 20.

    Gasiunas G. Barrangou R. Horvath P. Siksnys V., 2012 PNAS Plus: Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109: E2579–E2586.

    Not in our library10.1073/pnas.1208507109

21. 21.

    Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J. A., 2012 A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337: 816–821.

    Not in our library10.1126/science.1225829

22. 22.

    Katic I. Großhans H., 2013 Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics 195: 1173–1176.

    Not in our library10.1534/genetics.113.155754

23. 23.

    Kim H. Ishidate T. Ghanta K. S. Seth M. Conte D., 2014 A Co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics 197: 1069–1080.

    Not in our library10.1534/genetics.114.166389

24. 24.

    Kutscher L. M. Shaham S., 2014 Forward and reverse mutagenesis in C. elegans. WormBook 1–26.

    Not in our library10.1895/wormbook.1.167.1

25. 25.

    Lo T. W. Pickle C. S. Lin S. Ralston E. J. Gurling M., 2013 Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics 195: 331–348.

    Not in our library10.1534/genetics.113.155382

26. 26.

    Mahoney T. R. Luo S. Round E. K. Brauner M. Gottschalk A., 2008 Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 105: 16350–16355.

    Not in our library10.1073/pnas.0803617105

27. 27.

    Mali P. Yang L. Esvelt K. M. Aach J. Guell M., 2013 RNA-Guided Human Genome Engineering via Cas9. Science 339: 823–826.

    Not in our library10.1126/science.1232033

28. 28.

    Maruyama T. Dougan S. K. Truttmann M. C. Bilate A. M. Ingram J. R., 2015 Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33: 538–542.

    Not in our library10.1038/nbt.3190

29. 29.

    Mello C. Fire A., 1995 DNA transformation. Methods Cell Biol. 48: 451–482.

    Not in our library10.1016/s0091-679x(08)61399-0

30. 30.

    Norris A. D. Kim H. M. Colaiácovo M. P. Calarco J. A., 2015 Efficient genome editing in Caenorhabditis elegans with a toolkit of dual-marker selection cassettes. Genetics 201: 449–458.

    Not in our library10.1534/genetics.115.180679

31. 31.

    Paix A. Folkmann A. Rasoloson D. Seydoux G., 2015 High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics 201: 47–54.

    Not in our library10.1534/genetics.115.179382

32. 32.

    Paix A. Folkmann A. Seydoux G., 2017 Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans. Methods 121–122: 86–93.

    Not in our library10.1016/j.ymeth.2017.03.023

33. 33.

    Paquet D. Kwart D. Chen A. Sproul A. Jacob S., 2016 Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533: 125–129.

    Not in our library10.1038/nature17664

34. 34.

    Schwartz M. L. Jorgensen E. M., 2016 SapTrap, a toolkit for high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans. Genetics 202: 1277–1288.

    Not in our library10.1534/genetics.115.184275

35. 35.

    Thomas J. H., 1990 Genetic analysis of defecation in Caenorhabditis elegans. Genetics 124: 855–872.

    Not in our library10.1093/genetics/124.4.855

36. 36.

    Thompson O. Edgley M. Strasbourger P. Flibotte S. Ewing B., 2013 The million mutation project: a new approach to genetics in Caenorhabditis elegans. Genome Res. 23: 1749–1762.

    Not in our library10.1101/gr.157651.113

37. 37.

    Tzur Y. B. Friedland A. E. Nadarajan S. Church G. M. Calarco J. A., 2013 Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics 195: 1181–1185.

    Not in our library10.1534/genetics.113.156075

38. 38.

    Vallin E. Gallagher J. Granger L. Martin E. Belougne J., 2012 A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community. PLoS One 7: e30482

    Not in our library10.1371/journal.pone.0030482

39. 39.

    Waaijers S. Portegijs V. Kerver J. Lemmens B. B. L. G. Tijsterman M., 2013 CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans. Genetics 195: 1187–1191.

    Not in our library10.1534/genetics.113.156299

40. 40.

    Wang H. Girskis K. Janssen T. Chan J. P. Dasgupta K., 2013 Neuropeptide secreted from a pacemaker activates neurons to control a rhythmic behavior. Curr. Biol. 23: 746–754.

    Not in our library10.1016/j.cub.2013.03.049

Cited by papers we hold 1

• Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes. · 2022
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