Off-target genomic DNA sites with partial complementarity adjacent to Staphylococcus aureus Cas9 Protospacer Adjacent Motif (SaCas9 off-target sites)

Off-target genomic DNA sites with partial complementarity adjacent to Staphylococcus aureus Cas9 (SaCas9) Protospacer Adjacent Motif (PAM) represent unintended locations in the genome where the SaCas9 enzyme may bind and induce double-strand breaks. SaCas9 is a compact CRISPR-associated endonuclease frequently utilized in gene therapy due to its small size, which allows for efficient packaging into adeno-associated virus (AAV) delivery vectors [1]. While SaCas9 requires a specific NNGRRT PAM sequence to initiate DNA cleavage, the enzyme can tolerate several mismatches between the guide RNA (gRNA) and the genomic DNA, leading to cleavage at sites that resemble the intended target [2]. These unintended modifications pose significant safety risks in a clinical context, including the potential for chromosomal translocations, the activation of proto-oncogenes, or the disruption of essential tumor suppressor genes [3]. Consequently, these sites are not therapeutic targets but are instead critical safety liabilities that must be rigorously identified and minimized during the development of CRISPR-based medicines. Advanced genome-wide profiling techniques, such as GUIDE-seq and CIRCLE-seq, are employed to detect these off-target effects and ensure the precision of the gene-editing intervention [2]. [1] Ran, F. A., et al. (2015). Nature. [2] Kleinstiver, B. P., et al. (2015). Nature Biotechnology. [3] Tsai, S. Q., et al. (2015). Nature Biotechnology.

Other names

SaCas9 off-targetsNon-specific genomic cleavage sitesUnintended genomic modificationsSaCas9 off-target genomic loci

Unintended double-strand DNA breaks (DSBs) followed by error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) at genomic loci with sequence similarity to the intended target.