Predicting the mutations generated by repair of Cas9-induced double-strand breaks

Abstract

The DNA mutation produced by cellular repair of a CRISPR–Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR–Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

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Figure 1: Mutational profiles generated by CRISPR–Cas9 and a method for their high-throughput measurement.
Figure 2: Synthetic mutational profiles are reproducible, specific to individual gRNAs and closely resemble endogenously measured profiles in human K562 cells.
Figure 3: Mutational profiles are diverse and biased in K562 cells, as measured using 6,568 gRNAs with a median 991 sequenced reads with mutations per target.
Figure 4: Local sequence context strongly influences editing outcomes in the explorative set of gRNA–target pairs.
Figure 5: Differences between editing outcomes in K562-Cas9 and other cell lines and effector proteins.
Figure 6: Accurate prediction of repair profiles.

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Acknowledgements

We thank J. Eliasova for help with Figure 1, E. de Braekeleer from Wellcome Sanger Institute for providing the K562-Cas9 line, and A. Lawson for comments on the text. F.A. was supported by a Royal Commission for the Exhibition of 1851 Research Fellowship. L.P. was supported by Wellcome (206194) and the Estonian Research Council (IUT 34-4). H.P.H. was supported by a Wellcome Trust grant (200848/Z/16/Z) and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (100140). Y.G. is funded by Cancer Research UK C6/A18796 and Wellcome Trust Investigator Award 206388/Z/17/Z in the Jackson laboratory. F.M.M. was funded by a Marie Curie Intra-European Fellowship, project number 626375, DDR SYNVIA, and by Wellcome Trust Investigator Award 206388/Z/17/Z and an AstraZeneca Collaborative Award in the Jackson laboratory.

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F.A.: designed experiments, analyzed data, wrote paper. L.C.: designed experiments, performed experiments, wrote paper. C.A.: performed experiments in human iPSCs. A.J.S., E.M.: performed experiments in mouse ESCs. V. Kleshchevnikov: analyzed data, wrote paper. A.K., V. Kiselev: created web server. P.D.A., P.P.: performed experiments. M.K., A.R.B.: generated TREX2 constructs. H.H.: generated CHO-Cas9 line. Y.G., F.M.-M., S.P.J.: generated RPE-1-Cas9 and HAP1-Cas9 lines. L.P.: designed experiments, contributed to data analysis, wrote paper. All authors contributed to drafting the manuscript.

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Correspondence to Felicity Allen or Leopold Parts.

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Allen, F., Crepaldi, L., Alsinet, C. et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat Biotechnol 37, 64–72 (2019). https://doi.org/10.1038/nbt.4317

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