UP - logo
E-viri
Recenzirano Odprti dostop
  • Predictable and precise tem...
    Shen, Max W; Arbab, Mandana; Hsu, Jonathan Y; Worstell, Daniel; Culbertson, Sannie J; Krabbe, Olga; Cassa, Christopher A; Liu, David R; Gifford, David K; Sherwood, Richard I

    Nature, 11/2018, Letnik: 563, Številka: 7733
    Journal Article

    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.