Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5–11% of Cas9 guide RNAs targeting the human genome are ‘precise-50’, yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky–Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.
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High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database under accession codes SRP141261 and SRP141144. Processed data have been deposited under the following DOIs: https://doi.org/10.6084/m9.figshare.6838016, https://doi.org/10.6084/m9.figshare.6837959, https://doi.org/10.6084/m9.figshare.6837956, https://doi.org/10.6084/m9.figshare.6837953, and https://doi.org/10.6084/m9.figshare.6837947.
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The authors thank O. Juez, R. Jodhani and C. Araneo for technical assistance and the MIT Biomicro Center, the Harvard Medical School Biopolymers Facility, and the Broad Institute Genomics Platform for sequencing. The authors acknowledge funding from an NSF Graduate Research Fellowship to M.W.S.; an NWO Rubicon Fellowship to M.A.; 1R01HG010372 (C.A.C.); DARPA HR0011-17-2-0049, NIHRM1 HG009490, R01 EB022376, R35 GM118062, HHMI (D.R.L.); 1RO1HG008363, 1R01HG008754 (D.K.G.); 1K01DK101684, the Human Frontier Science Program, NWO, Brigham Research Institute, Harvard Stem Cell Institute, and American Cancer Society (R.I.S.).
Nature thanks D. Durocher, R. Platt and the anonymous reviewer(s) for their contribution to the peer review of this work.