title: Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9.
abstract: The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.
authors.affiliation: Harvard Medical School; Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Daniel.Bauer@childrens.harvard.edu.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
authors.affiliation: Harvard Medical School.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Canver
authors.affiliation: Harvard Medical School; Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Howard Hughes Medical Institute; stuart_orkin@dfci.harvard.edu.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA