Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking.
CRISPR-Cas Systems
Cell Line, Tumor
DNA
/ analysis
DNA Breaks, Double-Stranded
Gene Editing
Gene Knock-In Techniques
Gene Targeting
/ methods
Genes, Reporter
Genetic Engineering
HCT116 Cells
Homologous Recombination
Humans
Plasmids
/ metabolism
RNA, Guide, Kinetoplastida
/ genetics
Recombination, Genetic
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
19 11 2021
19 11 2021
Historique:
received:
19
05
2021
accepted:
08
11
2021
entrez:
20
11
2021
pubmed:
21
11
2021
medline:
8
3
2022
Statut:
epublish
Résumé
Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700-2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.
Identifiants
pubmed: 34799652
doi: 10.1038/s41598-021-01978-w
pii: 10.1038/s41598-021-01978-w
pmc: PMC8604973
doi:
Substances chimiques
RNA, Guide
0
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
22627Informations de copyright
© 2021. The Author(s).
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