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A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Kim H, Ishidate T, Ghanta KS, Seth M, Conte D, Shirayama M, Mello CC - Genetics (2014)

Bottom Line: Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans.The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols.Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.

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HR-mediated knock-in to generate fusion genes at endogenous loci. (A) Schematic of the Cas9/sgRNA target site and the donor plasmid for gfp::pie-1 knock-ins. The donor plasmid contains the gfp coding sequence inserted immediately after the start codon of pie-1, 1 kb of homology flanking the CRISPR-Cas9 cleavage site, and a silent mutation in the PAM site. (B) Strategy to screen for gfp knock-in lines. We placed three F1 rollers at a time on a 2% agar pad and screened for GFP expression using epifluorescence microscopy. GFP-expressing worms were individually recovered and allowed to make F2 progeny for 1 day before being lysed for PCR and DNA sequence analysis. We confirmed Mendelian inheritance of gfp knock-in alleles among F2 progeny. (C) GFP::PIE-1 expression in the germline of two- to four-cell embryos of gfp::pie-1 knock-in strains. (D) Immunoblot analysis showing PIE-1 expression levels in wild-type animals, MosSCI-mediated gfp::pie-1 knock-in animals, and CRISPR-Cas9-mediated gfp knock-in animals. A MosSCI strain of gfp::pie-1; pie-1(zu154) was obtained by crossing gfp::pie-1 (LGII) with the pie-1(zu154) (LGIII)  mutant. (E) mCherry expression in late embryos of the mCherry::vet-2 knock-in strain. (F) Schematic of Cas9/sgRNA target sequence, PAM site, and donor plasmid for pie-1::flag knock-in. The PAM is located in the last exon of pie-1. The donor plasmid includes flag coding sequence immediately before the pie-1 stop codon and ∼800-bp homology arms flanking the target site. (G) PCR and restriction analysis of an HR event. PCR products were generated using the primers indicated in F, and the products were digested with NheI. The pie-1::flag gene conversion introduces an NheI RFLP that is observed in F1 heterozygous and F2 homozygous pie-1::flag animals.
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fig3: HR-mediated knock-in to generate fusion genes at endogenous loci. (A) Schematic of the Cas9/sgRNA target site and the donor plasmid for gfp::pie-1 knock-ins. The donor plasmid contains the gfp coding sequence inserted immediately after the start codon of pie-1, 1 kb of homology flanking the CRISPR-Cas9 cleavage site, and a silent mutation in the PAM site. (B) Strategy to screen for gfp knock-in lines. We placed three F1 rollers at a time on a 2% agar pad and screened for GFP expression using epifluorescence microscopy. GFP-expressing worms were individually recovered and allowed to make F2 progeny for 1 day before being lysed for PCR and DNA sequence analysis. We confirmed Mendelian inheritance of gfp knock-in alleles among F2 progeny. (C) GFP::PIE-1 expression in the germline of two- to four-cell embryos of gfp::pie-1 knock-in strains. (D) Immunoblot analysis showing PIE-1 expression levels in wild-type animals, MosSCI-mediated gfp::pie-1 knock-in animals, and CRISPR-Cas9-mediated gfp knock-in animals. A MosSCI strain of gfp::pie-1; pie-1(zu154) was obtained by crossing gfp::pie-1 (LGII) with the pie-1(zu154) (LGIII) mutant. (E) mCherry expression in late embryos of the mCherry::vet-2 knock-in strain. (F) Schematic of Cas9/sgRNA target sequence, PAM site, and donor plasmid for pie-1::flag knock-in. The PAM is located in the last exon of pie-1. The donor plasmid includes flag coding sequence immediately before the pie-1 stop codon and ∼800-bp homology arms flanking the target site. (G) PCR and restriction analysis of an HR event. PCR products were generated using the primers indicated in F, and the products were digested with NheI. The pie-1::flag gene conversion introduces an NheI RFLP that is observed in F1 heterozygous and F2 homozygous pie-1::flag animals.

Mentions: To test this idea, we decided to use CRISPR-Cas9-mediated HR to introduce the gfp coding sequence immediately downstream of the start codon in the endogenous pie-1 locus (Figure 3A). The donor plasmid in this experiment contained NheI restriction sites flanking the gfp coding sequence, 1-kb homology arms, and a silent mutation that disrupts the PAM sequence at the sgRNA target site (Figure 3A). We generated three different donor constructs: gfp::pie-1(WT), gfp::pie-1(K68A), and gfp::pie-1(K68R). Each donor molecule was co-injected with vectors to express the sgRNA, Cas9, and rol-6 marker. We then directly examined the resulting F1 rolling animals for GFP::PIE-1 expression in the germline and embryos using epifluorescence microscopy (Figure 3B, see Materials and Methods). Using this approach, we obtained 9 independent gfp::pie-1(K68A) lines from 92 F1 rollers, 1 gfp::pie-1(K68R) line from 69 F1 rollers, and 1 gfp::pie-1(WT) line from 72 F1 rollers. Subsequent analyses revealed that each of these F1 animals was heterozygous for gfp::pie-1, and each strain incorporated both the gfp coding sequence and the PAM site mutation, as well as the linked K68A and K68R missense mutations. For unknown reasons, we found that one of the nine gfp::pie-1(K68A) lines could not be maintained.


A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.

Kim H, Ishidate T, Ghanta KS, Seth M, Conte D, Shirayama M, Mello CC - Genetics (2014)

HR-mediated knock-in to generate fusion genes at endogenous loci. (A) Schematic of the Cas9/sgRNA target site and the donor plasmid for gfp::pie-1 knock-ins. The donor plasmid contains the gfp coding sequence inserted immediately after the start codon of pie-1, 1 kb of homology flanking the CRISPR-Cas9 cleavage site, and a silent mutation in the PAM site. (B) Strategy to screen for gfp knock-in lines. We placed three F1 rollers at a time on a 2% agar pad and screened for GFP expression using epifluorescence microscopy. GFP-expressing worms were individually recovered and allowed to make F2 progeny for 1 day before being lysed for PCR and DNA sequence analysis. We confirmed Mendelian inheritance of gfp knock-in alleles among F2 progeny. (C) GFP::PIE-1 expression in the germline of two- to four-cell embryos of gfp::pie-1 knock-in strains. (D) Immunoblot analysis showing PIE-1 expression levels in wild-type animals, MosSCI-mediated gfp::pie-1 knock-in animals, and CRISPR-Cas9-mediated gfp knock-in animals. A MosSCI strain of gfp::pie-1; pie-1(zu154) was obtained by crossing gfp::pie-1 (LGII) with the pie-1(zu154) (LGIII)  mutant. (E) mCherry expression in late embryos of the mCherry::vet-2 knock-in strain. (F) Schematic of Cas9/sgRNA target sequence, PAM site, and donor plasmid for pie-1::flag knock-in. The PAM is located in the last exon of pie-1. The donor plasmid includes flag coding sequence immediately before the pie-1 stop codon and ∼800-bp homology arms flanking the target site. (G) PCR and restriction analysis of an HR event. PCR products were generated using the primers indicated in F, and the products were digested with NheI. The pie-1::flag gene conversion introduces an NheI RFLP that is observed in F1 heterozygous and F2 homozygous pie-1::flag animals.
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Related In: Results  -  Collection

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fig3: HR-mediated knock-in to generate fusion genes at endogenous loci. (A) Schematic of the Cas9/sgRNA target site and the donor plasmid for gfp::pie-1 knock-ins. The donor plasmid contains the gfp coding sequence inserted immediately after the start codon of pie-1, 1 kb of homology flanking the CRISPR-Cas9 cleavage site, and a silent mutation in the PAM site. (B) Strategy to screen for gfp knock-in lines. We placed three F1 rollers at a time on a 2% agar pad and screened for GFP expression using epifluorescence microscopy. GFP-expressing worms were individually recovered and allowed to make F2 progeny for 1 day before being lysed for PCR and DNA sequence analysis. We confirmed Mendelian inheritance of gfp knock-in alleles among F2 progeny. (C) GFP::PIE-1 expression in the germline of two- to four-cell embryos of gfp::pie-1 knock-in strains. (D) Immunoblot analysis showing PIE-1 expression levels in wild-type animals, MosSCI-mediated gfp::pie-1 knock-in animals, and CRISPR-Cas9-mediated gfp knock-in animals. A MosSCI strain of gfp::pie-1; pie-1(zu154) was obtained by crossing gfp::pie-1 (LGII) with the pie-1(zu154) (LGIII) mutant. (E) mCherry expression in late embryos of the mCherry::vet-2 knock-in strain. (F) Schematic of Cas9/sgRNA target sequence, PAM site, and donor plasmid for pie-1::flag knock-in. The PAM is located in the last exon of pie-1. The donor plasmid includes flag coding sequence immediately before the pie-1 stop codon and ∼800-bp homology arms flanking the target site. (G) PCR and restriction analysis of an HR event. PCR products were generated using the primers indicated in F, and the products were digested with NheI. The pie-1::flag gene conversion introduces an NheI RFLP that is observed in F1 heterozygous and F2 homozygous pie-1::flag animals.
Mentions: To test this idea, we decided to use CRISPR-Cas9-mediated HR to introduce the gfp coding sequence immediately downstream of the start codon in the endogenous pie-1 locus (Figure 3A). The donor plasmid in this experiment contained NheI restriction sites flanking the gfp coding sequence, 1-kb homology arms, and a silent mutation that disrupts the PAM sequence at the sgRNA target site (Figure 3A). We generated three different donor constructs: gfp::pie-1(WT), gfp::pie-1(K68A), and gfp::pie-1(K68R). Each donor molecule was co-injected with vectors to express the sgRNA, Cas9, and rol-6 marker. We then directly examined the resulting F1 rolling animals for GFP::PIE-1 expression in the germline and embryos using epifluorescence microscopy (Figure 3B, see Materials and Methods). Using this approach, we obtained 9 independent gfp::pie-1(K68A) lines from 92 F1 rollers, 1 gfp::pie-1(K68R) line from 69 F1 rollers, and 1 gfp::pie-1(WT) line from 72 F1 rollers. Subsequent analyses revealed that each of these F1 animals was heterozygous for gfp::pie-1, and each strain incorporated both the gfp coding sequence and the PAM site mutation, as well as the linked K68A and K68R missense mutations. For unknown reasons, we found that one of the nine gfp::pie-1(K68A) lines could not be maintained.

Bottom Line: Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans.The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols.Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.

Show MeSH
Related in: MedlinePlus