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Analyzing CRISPR genome-editing experiments with CRISPResso

2016; Nature Portfolio; Volume: 34; Issue: 7 Linguagem: Inglês

10.1038/nbt.3583

1546-1696

Luca Pinello, Matthew C. Canver, Megan D. Hoban, Stuart H. Orkin, Donald B. Kohn, Daniel E. Bauer, Guo‐Cheng Yuan,

RNA and protein synthesis mechanisms

Recent progress in genome editing technologies, in particular the CRISPR-Cas9 system, has provided new opportunities to investigate the biological functions of genomic sequences by targeted mutagenesis. Double strand breaks (DSBs) resulting from site-specific Cas9 cleavage can be resolved by endogenous DNA repair pathways such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). Deep sequencing of amplified genomic regions allows for quantitative and sensitive detection of targeted mutations. However, no standard analytic tool to date has been developed to systematically enumerate and visualize these events, and to solve challenging issues such as amplification or sequencing errors, experimental variation in sequence quality, ambiguous alignment of variable length indels, and difficulty in deconvoluting mixed HDR/NHEJ outcomes. To address these issues we developed CRISPResso, a robust computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites.