NDLI: Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9.

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Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Bauer, Daniel E. Canver, Matthew C. Orkin, Stuart H.
Description	Author Affiliation: Bauer DE ( Harvard Medical School); Canver MC ( Harvard Medical School.); Orkin SH ( Harvard Medical School)
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.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/52118
Journal	Journal of Visualized Experiments
Issue Number	95
Language	English
Publisher	MyJove Corp.
Publisher Date	2015-01-03
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Crispr-cas Systems Gene Deletion Genetic Engineering Animals Cell Line Clustered Regularly Interspaced Short Palindromic Repeats Genetics Dna Breaks, Double-stranded Dna End-joining Repair Rna, Guide Research Support, N.i.h., Extramural Video-audio Media
Content Type	Text
Resource Type	Article

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