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CRISPR Plasmids: C. elegans

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CRISPR Resources

The following CRISPR plasmids have been designed for use in C. elegans.

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

Plasmid Gene/Insert Promoter PI Publication Hidden Extra Search Info
Peft-3::cas9-SV40_NLS::tbb-2 3'UTRcodon optimized Cas9_SV40 NLS with intron (Synthetic)eef-1A.1 (eft-3) Calarco Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods. 2013 Jun 30. doi: 10.1038/nmeth.2532. pUC57
pDD162 (Peft-3::Cas9 + Empty sgRNA)Cas9 (Synthetic), Empty sgRNA (Other)eef-1A.1 (eft-3) , U6 Goldstein Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641. Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target site in the C. elegans genome. pCFJ601
SP6-hCas9-Ce-mRNAhCas9 with C. elegans 5' and 3' UTRs (Homo sapiens)SP6 Sternberg Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics. 2013 Aug 26. In vitro transcription of a mRNA encoding humanized Cas9 nuclease, with 3' UTRs suitable for germline expression in C. elegans, for doing CRISPR-Cas by RNA injection. Uses SP6 RNA polymerase. Bluescript
pIK86Cas9 (Synthetic)T7 Katic Targeted Heritable Mutation and Gene Conversion by Cas9-CRISPR in Caenorhabditis elegans. Genetics. 2013 Aug 26. C. elegans codon-optimized Cas9 with 2xSV40 NLS sequences. It has a T7 promoter for in vitro transcription, germline-compatible 5'UTR, tbb-2 3'UTR and a polyA sequence. pUC57
pMB62Cas9 (Synthetic)eef-1A.1 (eft-3) Boxem CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Genetics. 2013 Aug 26. Expresses C. elegans optimized Cas9::mEGFP from the eef-1A.1 (eft-3) promoter pBluescript SK+
pMB63Cas9 (Synthetic)eef-1A.1 (eft-3) Boxem CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Genetics. 2013 Aug 26. Expresses C. elegans optimized Cas9 from the eef-1A.1 (eft-3) promoter pBluescript SK+
pMB66Cas9 (Synthetic)hsp-16.48 Boxem CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Genetics. 2013 Aug 26. Expresses C. elegans optimized Cas9::mEGFP from the hsp-16.48 promoter pBluescript SK+
pMB67Cas9 (Synthetic)hsp-16.48 Boxem CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Genetics. 2013 Aug 26. Expresses C. elegans optimized Cas9 from the hsp-16.48 promoter pBluescript SK+
peft-3::cas-9::tbb-2 3'UTRcas9eef-1A.1 (eft-3) de Bono Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination. Nucleic Acids Res. 2013 Sep 5. this plasmid contains the codon optimized gene encoding Cas9 for C. elegans. pDEST
pJW1259Deletion Ward Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-conversion and Inactivation of NHEJ Repair. Genetics. 2014 Dec 9. pii: genetics.114.172361. Cas9 plasmid with sgRNA deleted; for use in C. elegans pJW1219
pME-Cas9codon optimized Cas9 (Other) Chen Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs. Genetics. 2015 Apr 8. pii: genetics.115.176917. expresses codon optimized Cas9 in zebrafish pME-MCS
AP56-5sgRNA for APa13 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
AP56-6sgRNA for APa13 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
AP56-7sgRNA for APa13 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
AP56-8sgRNA for APa13 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
AP54-3sgRNA for APs12 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
AP54-5sgRNA for APs12 (Caenorhabditis elegans)U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans fbf-2 gene pDD162
APq5271sgRNA for APa4-2U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting K08F4.2 in the middle of the ORF pDD162
APq5210sgRNA for APa4-2U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting K08F4.2 in the middle of the ORF pDD162
AP303-3sgRNA for APs1U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 5'end of C.elegans K08F4.2 pDD162
AP303-4sgRNA for APs1U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 5'end of C.elegans K08F4.2 pDD162
AP332-2sgRNA for APs4U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 5'end of C.elegans K08F4.2 pDD162
AP332-3sgRNA for APs4U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 5'end of C.elegans K08F4.2 pDD162
AP332-4sgRNA for APs4U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 5'end of C.elegans K08F4.2 pDD162
AP324-2sgRNA for APs6U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
AP324-3sgRNA for APs6U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
AP324-4sgRNA for APs6U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
AP334-2sgRNA for APs5U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
AP334-5sgRNA for APs5U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
AP334-6sgRNA for APs5U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans K08F4.2 pDD162
APCSD 53sgRNA for CSD 53U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans swan-2 pDD162
APCSD 54sgRNA for CSD 54U6 Seydoux Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics. 2014 Dec;198(4):1347-56. doi: 10.1534/genetics.114.170423. Epub 2014 Sep 23. co-expression of Cas9 and a sgRNA targeting 3'end of C.elegans swan-2 pDD162
AP568-2sgRNA for dpy‐10 Seydoux High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics. 2015 Sep;201(1):47-54. doi: 10.1534/genetics.115.179382. Epub 2015 Jul 17. co-expression of Cas9 and crRNA 589 target sequence pDD162
AP568-3sgRNA for dpy‐10 Seydoux High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics. 2015 Sep;201(1):47-54. doi: 10.1534/genetics.115.179382. Epub 2015 Jul 17. co-expression of Cas9 and crRNA 589 target sequence pDD162
pRB1080VQR Cas9 variant (Synthetic)eef-1A.1 (eft-3) Fire Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans. Genetics. 2015 Dec 17. pii: genetics.115.185041. Expresses VQR Cas9 variant with eef-1A.1 (eft-3) promoter pCFJ601
pRB1081dpy-10 gRNA (Synthetic)U6 Fire Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans. Genetics. 2015 Dec 17. pii: genetics.115.185041. Expresses gRNA for dpy-10 conversion using NGAG PAM and VQR Cas9 variant pRB1017
pRB1083VRER Cas9 variant (Synthetic)eft-3 (eef-1A.1) Fire Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans. Genetics. 2015 Dec 17. pii: genetics.115.185041. Expresses VRER Cas9 variant with eef-1A.1 (eft-3) promoter pCFJ601
pRB1084dpy-10 gRNA (Synthetic)U6 Fire Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans. Genetics. 2015 Dec 17. pii: genetics.115.185041. Expresses gRNA for dpy-10 conversion using NGCG PAM and VRER Cas9 mutant pRB1017
pUAS:Cas9T2AGFP;U6:sgRNA1;U6:sgRNA25x UAS (Saccharomyces cerevisiae), Cas9 (Other), T2A, GFP, U6 promoter (Danio rerio) Del Bene 2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 2016 Mar 8. Tissue-specific knock-out in zebrafish, Cas9 and GFP driven by a UAS promoter pminiTol2
pUAS:Cas9T2ACre;U6:sgRNA1;U6:sgRNA25xUAS, Cas9, Cre, U6 promoter Del Bene 2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 2016 Mar 8. Tissue-specific knock-out in zebrafish, Cas9 and Cre driven by a UAS promoter pminiTol2
pDD121Cas9 (Synthetic) Goldstein Unpublished SapTrap/SEC plasmids (unpublished) Cas9 expression in C. elegans pCFJ601
pCFJ150-Cas9(dpiRNA)Cas9(dpiRNA) (Caenorhabditis elegans)eft-3 Lee The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes. Science. 2018 Feb 2;359(6375):587-592. doi: 10.1126/science.aao2840. Epub 2018 Feb 1. eft-3p::Cas9(dpiRNA)::tbb-2 3'UTR construct used for MosSCI in nematode. Cas9 is optimized by removing all piRNA targeting sites to allow germline expression. pCFJ150
Ccas9LacZ Vellai Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research. Nucleic Acids Res. 2018 Jun 19. pii: 5040046. doi: 10.1093/nar/gky516. Enhanced C. elegans Cas9 vector. Ccas9LacZ contains a lacZ coding sequence, two Cas9 and two type-IIs restriction enzyme recognition sites next to the U6 promoter and gRNA sequences. pDD162 (Peft-3::Cas9 + Empty sgRNA, Addgene no.: #47549)

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is often limited to a single category (cut, nick, etc.) On the other hand, gRNA plasmids that do not co-express a Cas protein can be paired with a wide variety of Cas-containing plasmids.

gRNA Plasmid Promoter Cloning
Enzyme(s)		 Delivery Resistance Co-expressed Cas9 Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
pMB70 cU6 BsaI Transfection none, need Cas9 plasmid Boxem
pMB60 T7 BsaI In vitro transcription none, need Cas9 plasmid Boxem
PU6::klp-12_sgRNA cU6 PCR (see paper) Transfection none, need Cas9 plasmid Calarco
pDD162 (Peft-3::Cas9 + Empty sgRNA) R07E5.16 U6 yes, cut Goldstein
DR274 T7 BsaI In vitro transcription none, need Cas9 plasmid Joung
SP6-sgRNA-scaffold SP6 AflII In vitro transcription none, need Cas9 plasmid Sternberg

Do you have suggestions for other plasmids that should be added to this list?

Fill out our Suggest a Plasmid form or e-mail [email protected] to help us improve this resource!