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
Highlights
CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci
The selection criteria for an single-stranded oligodeoxynucleotide (ssODN) donor template begins with identifying the targeting strand (T strand; ssODN that is complementary to the CRISPR–Cas[9] guide RNA (gRNA)) or the non-targeting strand (NT strand; ssODN that is non-complementary to the gRNA and contains the ‘NGG’ protospacer adjacent motif (PAM) sequence) as driving the most efficient repair via homology-directed repair (HDR)
Donor ssODNs containing 40-nt homology arms were designed to insert a six base EcoRI restriction digest recognition site (‘GAATTC’) at the Cas[9] cleavage site which canonically lies three bases in the 5’ direction of the PAM, as illustrated in Fig. 1A. ssODNs were delivered to Jurkat and HAP1 cells along with their respective gRNAs as Cas[9] RNP complexes by nucleofection, and the editing frequencies were assessed by generation sequencing (NGS)
Summary
CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. Cas[9] nickases can be used with an individual guide to induce single DNA nicks and induce a repair pathway termed alternative-HDR15, 16 It is more common and often more efficient to perform genome editing at DSBs generated by using a nickase with a pair of gRNAs targeting opposite DNA strands in a “paired nicking strategy”[17]. Cas12a has non-specific single-stranded DNase (ssDNase) activity that is activated upon binding to the target DNA strand[29] This could potentially impact the ability of Cas12a to mediate efficient HDR if the ssODN is degraded before it is able to act as a donor template
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