Abstract
CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
Highlights
Foreign donor DNA can be integrated into a host genome using any of several host Double-strand breaks (DSBs) repair mechanisms
An advantage of homology-directed repair (HDR)-mediated integration over Non-homologous end joining (NHEJ)/microhomology-mediated end joining (MMEJ)-based methods is the availability of negative selection markers, which are located outside the donor DNA homology arms and are only expressed if the entire donor is integrated through NHEJ/MMEJ
We propose BiPoD, an HDR-mediated knock-in method comprising in vivo-linearization of donors, knockdown of Polq, and inhibition of DNA-PK by M3814
Summary
Foreign donor DNA can be integrated into a host genome using any of several host DSB repair mechanisms. Non-homologous end joining (NHEJ) is a major pathway for HDR-independent genomic integration, in which the ends of linearized donor DNA are directly ligated to the broken ends of genomic D NA3. This process is executed by multiple machineries, including DNA-dependent protein kinase (DNA-PK) and DNA ligase IV (LIG4)[4,5]. Donor DNA is efficiently integrated into CRISPR/Cas9-induced DSB sites (on-target) through NHEJ or MMEJ with or without indel m utation[9,10]. Combining in vivo-linearization of donor DNA, knockdown of the Polq gene, and chemical inhibition of DNA-PK, we achieved specific HDR-mediated knock-in with very high efficiency of biallelic integration. Our method, termed BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition), provides an easy and rapid way to obtain precise knock-in mESCs
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