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Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

By Hera Canaj, Jeffrey A Hussmann, Han Li, Kyle A. Beckman, Leeanne Goodrich, Nathan H Cho, Yucheng J Li, Daniel A Santos, Aaron McGeever, Edna M Stewart, Veronica Pessino, Mohammad A. Mandegar, Cindy Huang, Li Gan, Barbara Panning, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti

Posted 13 Nov 2019
bioRxiv DOI: 10.1101/841098

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

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