Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.

Keywords: CRISPR/Cas9 genome editing; Double-strand break (DSB) repair pathway choice; Target binding affinity; Target residence.

Fig. 1. Targeted binding of Cas9 to genomic DNA in the context of chromatin. Cas9 undergoes a conformational change in complex with sgRNA and binds a genomic target in the context of chromatin via several interactions, including the interaction of the PI domain of Cas9 with the PAM sequence of the target, the 20-nt Watson-Crick base pairing between the sgRNA spacer and the target strand, and non-specific interactions between Cas9 and target DNA. Two arginine residues (R1333 and R1335) in the PI domain directly contact the conserved GG dinucleotide in the PAM sequence. Epigenetic modifications, such as histone acetylation, histone methylation， and DNA methylation, affect Cas9-sgRNA targeting to DNA. Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9； NUC: the nuclease lobe; REC: the recognition lobe; PAM: protospacer adjacent motif; PI: PAM-interacting; sgRNA: single-guicle RNA.

Fig. 2. Distinct end configurations generated by different forms of Cas9-sgRNA dissociation from cleaved DNA. Spontaneous release of Cas9-sgRNA from a cleaved target generates a conventional two-ended DSB that can be directly recognized by core NHEJ factors. Persistent Cas9-sgRNA target binding increases the probability of encountering local DNA replication or transcription. Collision with a replication fork may generate a DSB with three ends: a blunt end at the leading strand, a 3’-overhanging end at the lagging strand, and a blunt end away from the replication fork. Transcription may also dislodge Cas9-sgRNA from the cleaved target site, forming a potentially unique end configuration. Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9; DNA-PKcs: DNA-dependent protein kinase catalytic subunit; DSB: double-strand break; NHEJ: non-homologous end joining; PAM: protospacer adjacent motif; RNAP: RNA polymerase; sgRNA: single-guide RNA.