Seed sequences mediate off-target activity in the CRISPR-interference (CRISPRi) system

The CRISPR interference (CRISPRi) system is a powerful tool that is widely used to selectively and efficiently silence genes in functional genomics research applications. However, the extent of off-target activity associated with the CRISPRi system has not been systematically investigated. Here, we utilized a genome-wide CRISPRi Cas9 single guide RNA (sgRNA) library to investigate the presence of off-target activity and its effects on gene expression. Our findings suggest that off-target effects in CRISPRi are quite pervasive and have direct and indirect impact on gene expression. Most of the identified off-target activities can be accounted for by complementarity of the PAM-proximal genomic sequence with the 3’ half of the sgRNA spacer sequence, the seed sequence. We also report that not all PAM proximal positions are equally important for binding of sgRNA to the DNA and that mismatches in the PAM-proximal regions can be tolerated depending on the sequence context.