ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/096/SRR27483796/SRR27483796.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/099/SRR27483799/SRR27483799.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/005/SRR27483805/SRR27483805.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/000/SRR27483800/SRR27483800.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/093/SRR27483793/SRR27483793.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/094/SRR27483794/SRR27483794.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/004/SRR27483804/SRR27483804.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/002/SRR27483802/SRR27483802.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/098/SRR27483798/SRR27483798.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/095/SRR27483795/SRR27483795.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/092/SRR27483792/SRR27483792.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/001/SRR27483801/SRR27483801.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/006/SRR27483806/SRR27483806.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/097/SRR27483797/SRR27483797.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR274/003/SRR27483803/SRR27483803.fastq.gzprimaryOK200GenomicsGenentechhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1063363Homo 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: RNAseq across two different cell lines, Jurkat and EA.hy926, infected with single guides for ADAT1, HMGB1, GRK4, and LRRC34 respectively, with controlsxref:PubMed:39510079ENACRISPR Locus, CRISPR Loci, CRISPR Spacer Sequences, CRISPR Clusters, CRISPR-Cas Loci, CRISPR Sequence, anatomical systems, Element, Clustered Regularly Interspaced Short Palindromic Repeat, Activity, Elements, CRISPR Spacer, CRISPR Arrays, CRISPR Array, RNA-seq., CRISPR-Cas, Clusters, CRISPR Spacer Sequence, Locus, CRISPR Sequences, CRISPR Cluster, CRISPR, CRISPRs, Sequences, CRISPR-Cas Locus, CRISPR Element, Cluster, Sequence, Loci, Spacers, CRISPR Elements, Array, CRISPR Cas Loci, Whole Transcriptome Shotgun Sequencing, Spacer Sequences, Spacer, General activity, Arrays, CRISPR Spacers, Spacer SequenceCRISPR Locus, CRISPR Loci, CRISPR Spacer Sequences, CRISPR Clusters, CRISPR-Cas Loci, CRISPR Sequence, anatomical systems, Element, Clustered Regularly Interspaced Short Palindromic Repeat, Activity, Elements, CRISPR Spacer, CRISPR Arrays, CRISPR Array, RNA-seq., CRISPR-Cas, Clusters, CRISPR Spacer Sequence, Locus, CRISPR Sequences, CRISPR Cluster, CRISPR, CRISPRs, Sequences, CRISPR-Cas Locus, CRISPR Element, Cluster, Sequence, Loci, Spacers, CRISPR Elements, Array, CRISPR Cas Loci, Whole Transcriptome Shotgun Sequencing, Spacer Sequences, Spacer, General activity, Arrays, CRISPR Spacers, Spacer SequencefalsePervasive off-target activity in CRISPR-interference (CRISPRi) system [RNA-seq]Pervasive off-target activity in CRISPR-interference (CRISPRi) system [RNA-seq]2025-09-242024-10-18PRJNA1063363GSE252977960639510079