Competitive Advantages
High efficiency in generating animal models, Improved knock-in efficiency, Low toxicity and low cost.
The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, can efficiently generate gene knockouts of variably sizes. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homologydirected-repair (HDR) pathway with CRISPR cleavage. HDR-mediated gene knock-in experiments are inefficient, with no reports of successful gene knock-in with DNA fragments larger than 4 kb. Targeted insertion of large DNA fragments (7.4 and 5 .8 kb) into the genomes of mouse embryonic stem cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors was performed and indicate that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRIS PR-mediated double-strand breaks (DSBs ), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected.For precise genome editing by inserting or deleting a designed fragment, gene targeting based on homologous recombination (HR) is often the preferred methodology. However, the efficiency of traditional gene targeting is generally low, ranging from undetectable to 0.1%. The breaks induced by CRISPR can also be repaired through the homology directed-repair (HDR) pathway. However, the efficiency of HDR-mediated knock-in is typically inefficient. Previous research indicated that HDR and NHEJ compete with each other for the same break site and thus the efficiency of HDR could be significantly increased by inhibiting NHEJ. Furthermore, it is unknown whether or not inhibiting NHEJ is effective for knocking in large fragments (>4 kb) containing multiple genes or a single large gene. Our inventors have addressed many of the shortcomings of the existing techniques with an innovative CRISPR/HDR-mediated knock-in procedure that demonstrates (i) the ability to utilize the more accurate HDR pathway without using any NHEJ inhibitors, (ii) knock-in efficiencies equivalent to previous efforts utilizing the NHEJ inhibitors; (iii) the ability to knock-in large fragments and (iv) the absence of undesirable bi-allelic knock-ins. These results demonstrate a significant improvement in accuracy, knock-in fragment size, and cost. This technology can be applied to the rapid generation of cellular and animal models, live imaging of cellular genomes, and functional genomic screens, among other applications.
