Predictable and precise template-free CRISPR editing of pathogenic variants

• Published in: Nature (London) Vol. 563; no. 7733; pp. 646 - 651
• Main Authors: 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.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2018; Nature Publishing Group
• Subjects: 45; 45/77; 631/114/2785; 631/208/191; Algorithms; Alleles; Artificial intelligence; Base Sequence; Cell lines; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; CRISPR-Cas technology; Data mining; Deoxyribonucleic acid; Design; Disruption; DNA; DNA repair; DNA Repair - genetics; Editing; Fibroblasts; Fibroblasts - metabolism; Fibroblasts - pathology; Gene disruption; Gene Editing - methods; Gene Editing - standards; Genes; Genome editing; Genomes; Genomics; Genotype & phenotype; Genotypes; HCT116 Cells; HEK293 Cells; Hermanski-Pudlak Syndrome - genetics; Hermanski-Pudlak Syndrome - pathology; Hermansky-Pudlak syndrome; Human genetic engineering; Human genome; Humanities and Social Sciences; Humans; Innovations; K562 Cells; Learning algorithms; Libraries; Machine Learning; Medical research; Menkes Kinky Hair Syndrome - genetics; Menkes Kinky Hair Syndrome - pathology; Menkes syndrome; Methods; multidisciplinary; Mutation; Repair; Reproducibility of Results; Science; Science (multidisciplinary); Stem cell research; Stem cells; Substrate Specificity; Templates, Genetic; United States; Microhomology-mediated End Joining (MMEJ); Low-density Lipoprotein Receptor (LDLR); gRNA Expression Plasmid; Hermansky-Pudlak Syndrome (HPS1); Production Editor
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-018-0686-x

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. The authors use a machine-learning algorithm to predict the spectrum of CRISPR–Cas9-nuclease-mediated DNA repair outcomes at human genomic target sites.