CRISPR Plasmids: C. elegans

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'UTR	codon 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-mRNA	hCas9 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
	pIK86	Cas9 (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
	pMB62	Cas9 (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+
	pMB63	Cas9 (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+
	pMB66	Cas9 (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+
	pMB67	Cas9 (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'UTR	cas9	eef-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
	pJW1259	Deletion		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-Cas9	codon 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
	AP56-5	sgRNA 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
	AP56-6	sgRNA 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
	AP56-7	sgRNA 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
	AP56-8	sgRNA 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
	AP54-3	sgRNA 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
	AP54-5	sgRNA 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
	APq5271	sgRNA for APa4-2	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 K08F4.2 in the middle of the ORF pDD162
	APq5210	sgRNA for APa4-2	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 K08F4.2 in the middle of the ORF pDD162
	AP303-3	sgRNA for APs1	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 5'end of C.elegans K08F4.2 pDD162
	AP303-4	sgRNA for APs1	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 5'end of C.elegans K08F4.2 pDD162
	AP332-2	sgRNA for APs4	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 5'end of C.elegans K08F4.2 pDD162
	AP332-3	sgRNA for APs4	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 5'end of C.elegans K08F4.2 pDD162
	AP332-4	sgRNA for APs4	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 5'end of C.elegans K08F4.2 pDD162
	AP324-2	sgRNA for APs6	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 K08F4.2 pDD162
	AP324-3	sgRNA for APs6	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 K08F4.2 pDD162
	AP324-4	sgRNA for APs6	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 K08F4.2 pDD162
	AP334-2	sgRNA for APs5	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 K08F4.2 pDD162
	AP334-5	sgRNA for APs5	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 K08F4.2 pDD162
	AP334-6	sgRNA for APs5	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 K08F4.2 pDD162
	APCSD 53	sgRNA for CSD 53	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 swan-2 pDD162
	APCSD 54	sgRNA for CSD 54	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 swan-2 pDD162
	AP568-2	sgRNA 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
	AP568-3	sgRNA 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
	pRB1080	VQR 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
	pRB1081	dpy-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
	pRB1083	VRER 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
	pRB1084	dpy-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:sgRNA2	5x 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:sgRNA2	5xUAS, 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
	pDD121	Cas9 (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