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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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“unc-22” Co-CRISPR as a marker to indicate actively expressed Cas9. (A) Schematic of Co-CRISPR strategy to identify functional sgRNAs targeting avr genes. sgRNAs targeting avr-14 and avr-15 were co-injected with a functional unc-22 sgRNA, the Cas9 expression vector, and the rol-6 transformation marker. F1 rollers or twitchers were transferred to individual plates. The plates were allowed to starve, and then they were copied to plates containing 2 ng/ml ivermectin to identify CRISPR-Cas9-induced avr-14; avr-15 double mutants. (B) Indel sequences in avr-14; unc-22; avr-15 triple mutants. avr-15 isolate 15 carried different indels on each allele. Sequences labeled with a question mark could not be precisely determined. (C) Comparison of twitcher-based indel frequency and roller-based indel frequency.
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fig2: “unc-22” Co-CRISPR as a marker to indicate actively expressed Cas9. (A) Schematic of Co-CRISPR strategy to identify functional sgRNAs targeting avr genes. sgRNAs targeting avr-14 and avr-15 were co-injected with a functional unc-22 sgRNA, the Cas9 expression vector, and the rol-6 transformation marker. F1 rollers or twitchers were transferred to individual plates. The plates were allowed to starve, and then they were copied to plates containing 2 ng/ml ivermectin to identify CRISPR-Cas9-induced avr-14; avr-15 double mutants. (B) Indel sequences in avr-14; unc-22; avr-15 triple mutants. avr-15 isolate 15 carried different indels on each allele. Sequences labeled with a question mark could not be precisely determined. (C) Comparison of twitcher-based indel frequency and roller-based indel frequency.

Mentions: DNA mixtures were micro-injected into the gonads of young adult worms. Plasmids for injection were prepared using a midiprep plasmid purification kit (Qiagen, no. 12143). For Co-CRISPR, we injected 50 ng/µl each vectors [Cas9 vector, unc-22 sgRNA vector (Co-CRISPR), two untested-sgRNAs, and pRF4::rol-6(su1006)] (Figure 2A). Micro-injection mixtures for HR contained 50 ng/μl each Cas9 vector, sgRNA vector, pRF4::rol-6(su1006), and HR donor vector. The final concentration of DNA in the injection mix did not exceed 200 ng/µl. For injection mixes with five different plasmids, 40 ng/µl of each plasmid was added. For HR experiments, we injected 40–60 worms and for disruptions, 20–30 worms. After recovering from injection, each worm was placed onto an individual plate.


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)

“unc-22” Co-CRISPR as a marker to indicate actively expressed Cas9. (A) Schematic of Co-CRISPR strategy to identify functional sgRNAs targeting avr genes. sgRNAs targeting avr-14 and avr-15 were co-injected with a functional unc-22 sgRNA, the Cas9 expression vector, and the rol-6 transformation marker. F1 rollers or twitchers were transferred to individual plates. The plates were allowed to starve, and then they were copied to plates containing 2 ng/ml ivermectin to identify CRISPR-Cas9-induced avr-14; avr-15 double mutants. (B) Indel sequences in avr-14; unc-22; avr-15 triple mutants. avr-15 isolate 15 carried different indels on each allele. Sequences labeled with a question mark could not be precisely determined. (C) Comparison of twitcher-based indel frequency and roller-based indel frequency.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: “unc-22” Co-CRISPR as a marker to indicate actively expressed Cas9. (A) Schematic of Co-CRISPR strategy to identify functional sgRNAs targeting avr genes. sgRNAs targeting avr-14 and avr-15 were co-injected with a functional unc-22 sgRNA, the Cas9 expression vector, and the rol-6 transformation marker. F1 rollers or twitchers were transferred to individual plates. The plates were allowed to starve, and then they were copied to plates containing 2 ng/ml ivermectin to identify CRISPR-Cas9-induced avr-14; avr-15 double mutants. (B) Indel sequences in avr-14; unc-22; avr-15 triple mutants. avr-15 isolate 15 carried different indels on each allele. Sequences labeled with a question mark could not be precisely determined. (C) Comparison of twitcher-based indel frequency and roller-based indel frequency.
Mentions: DNA mixtures were micro-injected into the gonads of young adult worms. Plasmids for injection were prepared using a midiprep plasmid purification kit (Qiagen, no. 12143). For Co-CRISPR, we injected 50 ng/µl each vectors [Cas9 vector, unc-22 sgRNA vector (Co-CRISPR), two untested-sgRNAs, and pRF4::rol-6(su1006)] (Figure 2A). Micro-injection mixtures for HR contained 50 ng/μl each Cas9 vector, sgRNA vector, pRF4::rol-6(su1006), and HR donor vector. The final concentration of DNA in the injection mix did not exceed 200 ng/µl. For injection mixes with five different plasmids, 40 ng/µl of each plasmid was added. For HR experiments, we injected 40–60 worms and for disruptions, 20–30 worms. After recovering from injection, each worm was placed onto an individual plate.

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