{"id":"doi:10.1186/s13059-017-1164-8","kind":"article","title":"Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage","authors":["Jianping Zhang","Xiaolan Li","Guohua Li","Wanqiu Chen","Cameron Arakaki","Gary D. Botimer","David J. Baylink","Lu Zhang","Wei Wen","Ya-Wen Fu","Jing Xu","Noah Chun","Weiping Yuan","Tao Cheng","Xiao‐Bing Zhang"],"author_count":15,"year":2017,"published":"2017-02-20","venue":"Genome biology","cited_by":480,"doi":"10.1186/s13059-017-1164-8","arxiv_id":null,"nct_id":null,"source_url":"https://doi.org/10.1186/s13059-017-1164-8","archive_url":"https://space-kitty.com/papers/doi:10.1186/s13059-017-1164-8","updated":null,"pmid":"28219395","pmcid":null,"openalex_id":"W2590493137","pdf_url":"https://genomebiology.biomedcentral.com/track/pdf/10.1186/s13059-017-1164-8","abstract":"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.","abstract_license":"CC-BY (publisher license, via OpenAlex)","license":"cc-by","is_open_access":true,"retracted":false,"peer_reviewed":null,"categories":["CRISPR and Genetic Engineering"],"details":null,"provenance":"openalex","attribution":"Abstract of \"Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage\" by Jianping Zhang, Xiaolan Li, Guohua Li et al., https://doi.org/10.1186/s13059-017-1164-8, used under its Creative Commons Attribution license (CC-BY); markup removed, otherwise unmodified.","topics":[{"slug":"gene-editing-therapies","name":"CRISPR & Gene-Editing Therapies","domain_slug":"medicine"}],"cited_in_dossiers":[]}