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

Canaj, H.; Hussmann, J. A.; Li, H.; Beckman, K. A.; Goodrich, L.; Cho, N. H.; Li, Y. J.; Santos, D. A.; McGeever, A.; Stewart, E. M.; Pessino, V.; Mandegar, M. A.; Huang, C.; Gan, L.; Panning, B.; Huang, B.; Weissman, J. S.; Leonetti, M. D.

2019-11-13 genomics
10.1101/841098 bioRxiv
Show abstract

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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