Journal
GENOME BIOLOGY
Volume 18, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/s13059-017-1164-8
Keywords
CRISPR; Genome editing; Knockin; Homology-directed repair (HDR); Non-homologous end joining (NHEJ); Donor design; 293 T; Human induced pluripotent stem cells
Funding
- Ministry of Science and Technology of China [2015CB964902, 2013CB966902, 2012CB966601]
- National Natural Science Foundation of China [81500148, 81570164, 81421002]
- Loma Linda University School of Medicine GCAT grant
- Telemedicine and Advanced Technology Research Center [W81XWH-08-1-0697]
Ask authors/readers for more resources
Background: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). Results: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97-100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs. Conclusions: Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available