Abstract
The CRISPR interference (CRISPRi) system is a powerful tool for selectively and efficiently silencing genes in functional genomics research applications. However, its off-target activity 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 impacts on gene expression. Most of the identified off-targets can be accounted for by complementarity of the protospacer adjacent motif (PAM)-proximal genomic sequence with the 3' half of the sgRNA spacer sequence, the seed sequence. We also report that while the stability of off-target binding is primarily driven by the PAM-proximal seed sequences, variations in the length of these seed sequences and the degree of mismatch tolerance at various positions can differ across different sgRNAs.