On-target Knockin of Large Cons tructs into TheMouse Genome via Combining CRISPR/Cas۹ and Nocodazole

On-target integration of large cassettes via homolog-directedrepair (HDR) has several applications. The knock-in efficiencyof large cons tructs is a challenging issue in mammalian genome.Nocodazole is an antineoplas tic agent that induces arresting with cell cycle G۲/M-phase. At this phase the highes trate of homology directed repair (HDR) is expected comparedto the insertions/deletions (indels) rate. In this s tudy, we madeseveral large cons tructs (۱۲.۰-۱۳.۰ kb) which included theCRISPR/Cas۹ sys tem as part of the large DNA donor (۵.۰-۷.۰kb) harbored by two homology arms specific for the promoterregion of the Venus transgene. In this sys tem, random indelsdirected by the CRISPR/Cas۹ sys tem did not knock out the Venusexpression. However, only insertion of the CRISPR/Cas۹-contained cons tructs into the promoter region could induce theVenus knockout. We used this s trategy to evaluate targeted integrationefficiency into a transgenic murine embryonic fibroblas t(MEF) cell line carrying a single copy of the Venus transgene.We es tablished a detection assay by which HDR events couldbe discriminated from the error-prone non-homologous end joining (NHEJ) events. Cell pre-treatment with nocodazole didnot affect the integration rate. However, direct inclusion of nocodazoleinto the electroporation medium could significantly improvethe HDR rate, on average ۲۴ % efficiency. In conclusion,the results of this s tudy showed that using cell synchronizationreagents in the electroporation medium can efficiently induceHDR rate in the mammalian genome.

کلیدواژه ها:

Cell Synchronization ، CRISPR/Cas۹ ، HDR ، LargeCons tructs ، Nocodazole