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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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A blasticidin-resistance marker to select pie-1 knockout mutants. (A) Schematic of the Cas9/sgRNA target sequence and an HR donor plasmid in which a heterologous blasticidin-resistance (BSD) gene replaces a region of pie-1 and is flanked by 1-kb homology arms. The BSD gene is under the control of the rpl-28 promoter (568 bp) and 3′-UTR (568 bp). (B) Schematic of the blasticidin selection strategy to precisely delete the pie-1 gene. pie-1a sgRNA was co-injected with the Cas9 expression vector, the rol-6 transformation marker, the pie-1∆::BSD donor construct, and the pCCM416::Pmyo-2::avr-15(+) counterselection vector. The indicated number of F1 rollers was transferred to the plates containing 2 ng/ml ivermectin to select against the extrachromosomal array and 100 μg/ml blasticidin to identify BSD knock-in lines. We identified two plates with resistant, fertile adults among 14 plates, 3–4 days after transferring animals.
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fig4: A blasticidin-resistance marker to select pie-1 knockout mutants. (A) Schematic of the Cas9/sgRNA target sequence and an HR donor plasmid in which a heterologous blasticidin-resistance (BSD) gene replaces a region of pie-1 and is flanked by 1-kb homology arms. The BSD gene is under the control of the rpl-28 promoter (568 bp) and 3′-UTR (568 bp). (B) Schematic of the blasticidin selection strategy to precisely delete the pie-1 gene. pie-1a sgRNA was co-injected with the Cas9 expression vector, the rol-6 transformation marker, the pie-1∆::BSD donor construct, and the pCCM416::Pmyo-2::avr-15(+) counterselection vector. The indicated number of F1 rollers was transferred to the plates containing 2 ng/ml ivermectin to select against the extrachromosomal array and 100 μg/ml blasticidin to identify BSD knock-in lines. We identified two plates with resistant, fertile adults among 14 plates, 3–4 days after transferring animals.

Mentions: Four days after injection, gravid F1 rolling adults were placed in groups of 10–15 animals per plate onto media containing ivermectin and blasticidin (Figure 4B). After 3–4 more days, the plates were scored for viable, fertile progeny. Insertion of BSD at the target locus was then confirmed by PCR and DNA sequencing (as described above). The total time from injection to recovery of HR events was 7–10 days. Although slightly longer in duration this procedure required ∼10 times less labor as only the relatively rare viable populations were subject to PCR and DNA sequence analysis. For donor molecules containing the Cbr-unc-119(+) selection, the procedure was essentially the same; however, blasticidin was omitted from the selective media and the recipient strain was both Cbr-unc-119 mutant and ivermectin resistant. Primers for screening HR events are listed in Table S3.


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)

A blasticidin-resistance marker to select pie-1 knockout mutants. (A) Schematic of the Cas9/sgRNA target sequence and an HR donor plasmid in which a heterologous blasticidin-resistance (BSD) gene replaces a region of pie-1 and is flanked by 1-kb homology arms. The BSD gene is under the control of the rpl-28 promoter (568 bp) and 3′-UTR (568 bp). (B) Schematic of the blasticidin selection strategy to precisely delete the pie-1 gene. pie-1a sgRNA was co-injected with the Cas9 expression vector, the rol-6 transformation marker, the pie-1∆::BSD donor construct, and the pCCM416::Pmyo-2::avr-15(+) counterselection vector. The indicated number of F1 rollers was transferred to the plates containing 2 ng/ml ivermectin to select against the extrachromosomal array and 100 μg/ml blasticidin to identify BSD knock-in lines. We identified two plates with resistant, fertile adults among 14 plates, 3–4 days after transferring animals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125384&req=5

fig4: A blasticidin-resistance marker to select pie-1 knockout mutants. (A) Schematic of the Cas9/sgRNA target sequence and an HR donor plasmid in which a heterologous blasticidin-resistance (BSD) gene replaces a region of pie-1 and is flanked by 1-kb homology arms. The BSD gene is under the control of the rpl-28 promoter (568 bp) and 3′-UTR (568 bp). (B) Schematic of the blasticidin selection strategy to precisely delete the pie-1 gene. pie-1a sgRNA was co-injected with the Cas9 expression vector, the rol-6 transformation marker, the pie-1∆::BSD donor construct, and the pCCM416::Pmyo-2::avr-15(+) counterselection vector. The indicated number of F1 rollers was transferred to the plates containing 2 ng/ml ivermectin to select against the extrachromosomal array and 100 μg/ml blasticidin to identify BSD knock-in lines. We identified two plates with resistant, fertile adults among 14 plates, 3–4 days after transferring animals.
Mentions: Four days after injection, gravid F1 rolling adults were placed in groups of 10–15 animals per plate onto media containing ivermectin and blasticidin (Figure 4B). After 3–4 more days, the plates were scored for viable, fertile progeny. Insertion of BSD at the target locus was then confirmed by PCR and DNA sequencing (as described above). The total time from injection to recovery of HR events was 7–10 days. Although slightly longer in duration this procedure required ∼10 times less labor as only the relatively rare viable populations were subject to PCR and DNA sequence analysis. For donor molecules containing the Cbr-unc-119(+) selection, the procedure was essentially the same; however, blasticidin was omitted from the selective media and the recipient strain was both Cbr-unc-119 mutant and ivermectin resistant. Primers for screening HR events are listed in Table S3.

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