BioProject

CRISPR/Cas proteins can be used to introduce double-stranded breaks (DSBs) at targeted genomic loci. More...

CRISPR/Cas proteins can be used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a DNA template during repair allows researchers to precisely introduce a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less precise NHEJ-mediated repair. Here, we describe design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes with synthetic guide RNAs. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and our findings provide design recommendations for each nuclease. Many of these findings have been implemented in the Alt-R HDR Design Tool, a novel bioinformatics tool for HDR donor template design. Additionally, we employ chemically modified Alt-R HDR Donor Oligos and Alt-R HDR Enhancers to boost HDR efficiency up to 9.6-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Less...