ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/067/SRR27488867/SRR27488867.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/070/SRR27488870/SRR27488870.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/068/SRR27488868/SRR27488868.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/066/SRR27488866/SRR27488866.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/072/SRR27488872/SRR27488872.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/071/SRR27488871/SRR27488871.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/069/SRR27488869/SRR27488869.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/065/SRR27488865/SRR27488865.fastq.gzprimary200OKGenomicsGenentechhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1063362Homo sapiensCRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems. Overall design: ChIPseq across two different cell lines, Jurkat and EA.hy926, infected with single guides for ADAT1, HMGB1, GRK4, and LRRC34 respectively and input controlsENAfalsePervasive off-target activity in CRISPR-interference (CRISPRi) system [ChIP-seq]Pervasive off-target activity in CRISPR-interference (CRISPRi) system [ChIP-seq]2025-09-242024-10-18PRJNA1063362GSE2529789606