Tandem Paired Nicking Promotes Precise Genome Editing with Scarce Interference by p53.

Cell Rep 2020 Jan;30(4):1195-1207.e7

Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan. Electronic address:

Targeted knockin mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knockin. Here, we demonstrate that tandem paired nicking, a method for targeted knockin involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knockin largely equivalent to that of the Cas9-nuclease-based approach. Tandem paired nicking seems to accomplish targeted knockin by DNA recombination analogous to Holliday's model and creates intended genomic changes without introducing additional nucleotide changes, such as silent mutations. Targeted knockin through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.12.064DOI Listing
January 2020
7.207 Impact Factor

Publication Analysis

Top Keywords

targeted knockin
24
tandem paired
20
paired nicking
20
efficiency targeted
8
knockin
6
targeted
6
tandem
5
nicking
5
nicks homologous
4
create nicks
4
homologous regions
4
mutations targeted
4
silent mutations
4
genome strand
4
strand scarcely
4
dna genome
4
donor dna
4
regions donor
4
intended genomic
4
knockin tandem
4

Altmetric Statistics

Similar Publications