Abstract
CRISPR–Cas proteins are RNA-guided nucleases 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 an exogenous DNA template during repair allows for the intentional, precise incorporation 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 (RNP) complexes. 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. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.