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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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Efficient CRISPR-Cas9-mediated gene disruption in transgenic animals. (A) Schematic of screen for CRISPR-Cas9 genome editing events. The dominant transformation marker rol-6 was co-injected with Cas9, pie-1a sgRNA, and donor plasmids. F1 rollers were screened for NHEJ-mediated indels by DNA sequencing. Among 93 F1 rollers, 22 indels were obtained. (B) Sequences of the wild-type pie-1 target site (top) and CRISPR-Cas9-mediated indels among F1 animals: (i) pie-1 homozygotes carrying the same indel on both alleles; (ii) pie-1 homozygotes carrying a different indel on each allele; and (iii) pie-1 heterozygotes. Lowercase letters indicate insertions, and dashes indicate deletions. The PAM is marked in red, and target sequences are marked in blue. The number of deleted (−) or inserted (+) bases is indicated to the right of each indel. The numbers in parentheses in (iii) represent the number of animals with the indels shown.
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fig1: Efficient CRISPR-Cas9-mediated gene disruption in transgenic animals. (A) Schematic of screen for CRISPR-Cas9 genome editing events. The dominant transformation marker rol-6 was co-injected with Cas9, pie-1a sgRNA, and donor plasmids. F1 rollers were screened for NHEJ-mediated indels by DNA sequencing. Among 93 F1 rollers, 22 indels were obtained. (B) Sequences of the wild-type pie-1 target site (top) and CRISPR-Cas9-mediated indels among F1 animals: (i) pie-1 homozygotes carrying the same indel on both alleles; (ii) pie-1 homozygotes carrying a different indel on each allele; and (iii) pie-1 heterozygotes. Lowercase letters indicate insertions, and dashes indicate deletions. The PAM is marked in red, and target sequences are marked in blue. The number of deleted (−) or inserted (+) bases is indicated to the right of each indel. The numbers in parentheses in (iii) represent the number of animals with the indels shown.

Mentions: While conducting CRISPR-Cas9 experiments to induce mutations in the pie-1 gene, we used the dominant co-injection marker rol-6 to monitor injection quality. From 60 injected animals, we obtained 93 fertile F1 rollers. Remarkably, we noted that several of these F1 rollers (5/93) produced 100% dead embryos exhibiting the distinctive pie-1 maternal-effect embryonic lethal phenotype (Mello et al. 1996) (Figure 1A). Genomic sequencing of these F1 adults identified lesions in the pie-1 gene consistent with Cas9-directed cleavage (Figure 1B). In some cases the maternal and paternal alleles exhibited distinct lesions, while in others, the same lesion was found in both alleles (Figure 1B). Since the DNA was delivered into the ovary of an adult, after the switch from sperm to oocyte development, the paternal allele must have been targeted in the F1 zygote soon after fertilization. The fact that both alleles exhibit identical lesions in some animals suggests that a chromosome previously cut by Cas9 and repaired by non-homologous end joining (NHEJ) was used as a donor molecule to copy the lesion into the homolog.


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)

Efficient CRISPR-Cas9-mediated gene disruption in transgenic animals. (A) Schematic of screen for CRISPR-Cas9 genome editing events. The dominant transformation marker rol-6 was co-injected with Cas9, pie-1a sgRNA, and donor plasmids. F1 rollers were screened for NHEJ-mediated indels by DNA sequencing. Among 93 F1 rollers, 22 indels were obtained. (B) Sequences of the wild-type pie-1 target site (top) and CRISPR-Cas9-mediated indels among F1 animals: (i) pie-1 homozygotes carrying the same indel on both alleles; (ii) pie-1 homozygotes carrying a different indel on each allele; and (iii) pie-1 heterozygotes. Lowercase letters indicate insertions, and dashes indicate deletions. The PAM is marked in red, and target sequences are marked in blue. The number of deleted (−) or inserted (+) bases is indicated to the right of each indel. The numbers in parentheses in (iii) represent the number of animals with the indels shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125384&req=5

fig1: Efficient CRISPR-Cas9-mediated gene disruption in transgenic animals. (A) Schematic of screen for CRISPR-Cas9 genome editing events. The dominant transformation marker rol-6 was co-injected with Cas9, pie-1a sgRNA, and donor plasmids. F1 rollers were screened for NHEJ-mediated indels by DNA sequencing. Among 93 F1 rollers, 22 indels were obtained. (B) Sequences of the wild-type pie-1 target site (top) and CRISPR-Cas9-mediated indels among F1 animals: (i) pie-1 homozygotes carrying the same indel on both alleles; (ii) pie-1 homozygotes carrying a different indel on each allele; and (iii) pie-1 heterozygotes. Lowercase letters indicate insertions, and dashes indicate deletions. The PAM is marked in red, and target sequences are marked in blue. The number of deleted (−) or inserted (+) bases is indicated to the right of each indel. The numbers in parentheses in (iii) represent the number of animals with the indels shown.
Mentions: While conducting CRISPR-Cas9 experiments to induce mutations in the pie-1 gene, we used the dominant co-injection marker rol-6 to monitor injection quality. From 60 injected animals, we obtained 93 fertile F1 rollers. Remarkably, we noted that several of these F1 rollers (5/93) produced 100% dead embryos exhibiting the distinctive pie-1 maternal-effect embryonic lethal phenotype (Mello et al. 1996) (Figure 1A). Genomic sequencing of these F1 adults identified lesions in the pie-1 gene consistent with Cas9-directed cleavage (Figure 1B). In some cases the maternal and paternal alleles exhibited distinct lesions, while in others, the same lesion was found in both alleles (Figure 1B). Since the DNA was delivered into the ovary of an adult, after the switch from sperm to oocyte development, the paternal allele must have been targeted in the F1 zygote soon after fertilization. The fact that both alleles exhibit identical lesions in some animals suggests that a chromosome previously cut by Cas9 and repaired by non-homologous end joining (NHEJ) was used as a donor molecule to copy the lesion into the homolog.

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