Improved Methods and Optimized Design for CRISPR Cas9 and Cas12a Homology-Directed Repair

CRISPR-Cas proteins are 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 for precise introduction of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive 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 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 were implemented in a novel online tool for HDR donor template design. Additionally, we employ chemically modified HDR donor templates in combination with a small molecule to boost HDR efficiency up to 10-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: [www.idtdna.com/HDR][1] ### Competing Interest Statement This work was supported by internal funds from Integrated DNA Technologies, Inc. The authors are employed by Integrated DNA Technologies, Inc., (IDT) which offers reagents for sale similar to some of the compounds described in the manuscript. GRR owns equity in DHR, the parent company of IDT. [1]: http://www.idtdna.com/HDR