We show that inactivation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) results in a drastic increase in efficiency of precise genome editing with CRISPR enzymes in human stem cells, allowing up to 79% of chromosomes to carry an intended nucleotide substitution when a single genomic site is targeted. When three different genes are simultaneously targeted, 12% of the isolated cells carry the targeted amino acid-changing substitutions in homozygous forms. These substitutions represent the first step towards resurrecting the proteome ancestral to Neandertals and modern humans. DNA-PKcs inactivation will greatly facilitate multiplexed precise genome editing in animal cells.

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