Biochemistry & Molecular Pharmacology
Program in Molecular Medicine; RNA Therapeutics Institute
Medical Subject Headings
Animals; Base Sequence; CRISPR-Associated Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Deoxyribonucleases; Genetic Markers; *Genome, Helminth; Homologous Recombination; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids; Sequence Analysis, DNA
Computational Biology | Genomics | Molecular Biology | Molecular Genetics
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. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. 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.
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Citation: Genetics. 2014 Aug;197(4):1069-80. doi: 10.1534/genetics.114.166389. Epub 2014 May 30. Link to article on publisher's site
CRISPR-Cas9 system, Co-CRISPR, CRISPR-Cas9-mediated HR, CRISPR-Cas9-induced indels, blasticidin selection
Kim, Heesun; Ishidate, Takao; Ghanta, Krishna S.; Seth, Meetu; Conte, Darryl Jr.; Shirayama, Masaki; and Mello, Craig C., "A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans" (2014). GSBS Student Publications. 1893.