Predictable and precise template-free CRISPR editing of pathogenic variants.

Following Cas9cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9editing 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 RNAspaired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60- base-pairdeletions and 1- base-pairinsertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5–11% of Cas9 guide RNAstargeting 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 syndromeand Menkes disease. This study establishes an approach for precise, template-free genome editing.