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A Systematic Analysis of the CRISPR/Cas9 Genome-editing Tool in Mammalian Cells♦

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells
    Open AccessPublished:August 01, 2014DOI:https://doi.org/10.1074/jbc.P114.564625
        Figure thumbnail gr1
        Deletion frequency inversely related to deletion size. Genomic deletion sizes ranged from 1.3 to 1026 kb, which show a decrease in the frequency of deletion events as deletion size increases.
        ♦ See referenced article, J. Biol. Chem. 2014, 289, 21312–21324
        The CRISPR/Cas9 method has quickly become popular for editing genes in a variety of model systems and organisms. The CRISPR acronym derives from clustered regularly interspaced palindromic repeats found in bacterial DNA; Cas9 is an RNA-guided endonuclease that makes double-stranded DNA breaks (DSBs). Although the system can be used to generate targeted single DSBs to either produce frameshift mutations in coding sequences or to stimulate homology-directed repair from an extrachromosomal template, a variation on the system relies on pairs of DSBs to produce larger genomic deletions. However, there hasn't been a systematic analysis of the system's efficiency to introduce these deletions at specified regions of DNA. In this Paper of the Week, a team led by Stuart H. Orkin at Harvard Medical School and the Howard Hughes Medical Institute developed an approach to create deletions in mammalian cells that ranged from 1.3 kb to more than 1 Mb. The investigators found that CRISPR/Cas9 does reliably produce the expected deletions. The frequency with which the system produced the deletion was inversely related to the size of the deletion. Orkin and colleagues concluded, “CRISPR/Cas9-mediated genomic deletion represents a robust method for loss-of-function studies in mammalian cells.”