Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Chen, X.Y.; Tasca, F.; Wang, Q.; Liu, J.; Janssen, J.M.; Brescia, M.D.; Bellin, M.; Szuhai, K.; Kenrick, J.; Frock, R.L.; Goncalves, M.A.F.V.

doi:doi:10.1093/nar/gkz1121

Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein...Show moreGenome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.Show less

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Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking