Improved methods and reagents for CRISPR homology-directed repair

CRISPR-Cas proteins introduce double-stranded breaks (DSBs) at targeted genomic loci and these are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a ssDNA template during repair allows researchers to precisely introduce a desired mutation by utilizing the HDR pathway. However, rates of HDR are often low compared to NHEJ-mediated repair. Here, we describe methods to improve the rate of HDR vs NHEJ-mediated repair by careful selection of template characteristics, including homology arm lengths, placement of synonymous SNPs in the HDR template, and the addition of chemical modifications to increase DNA stability against cellular nucleases. In addition, we demonstrate improved HDR rates when using Alt-R HDR Enhancer, a small molecule compound that increases the rate of HDR by inhibiting NHEJ-mediated repair. Increased HDR rates have been achieved in various cell lines, including iPSCs. Furthermore, we implement our findings in the Alt-R CRISPR HDR Design Tool, a novel bioinformatics tool for ssDNA HDR template design. The Alt-R CRISPR HDR Design Tool supports single-stranded designs up to 3 kb, single- and dual-guide designs (i.e., for use with Cas9-Nickases), guide selection suggestions, insertion of synonymous SNPs in coding regions, and more.