Off-target effects of CRISPRa on interleukin-6 expression

Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.

[1]  P. Fraser,et al.  Long-range enhancer–promoter contacts in gene expression control , 2019, Nature Reviews Genetics.

[2]  Jinke Wang,et al.  Gene activation by a CRISPR-assisted trans enhancer , 2019, eLife.

[3]  R. McPherson,et al.  Regulation of MFGE8 by the intergenic coronary artery disease locus on 15q26.1. , 2019, Atherosclerosis.

[4]  M. Borad,et al.  Hepatocytes direct the formation of a pro-metastatic niche in the liver. , 2019, Nature.

[5]  G. Lozano,et al.  CRISPR/Cas9 can mediate high-efficiency off-target mutations in mice in vivo , 2018, Cell Death & Disease.

[6]  G. B. Robb,et al.  Staphylococcus aureus Cas9 is a multiple-turnover enzyme , 2018, bioRxiv.

[7]  Jesse M. Engreitz,et al.  Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood , 2017, Nature.

[8]  P. D. Donohoue,et al.  NmeCas9 is an intrinsically high-fidelity genome-editing platform , 2017, Genome Biology.

[9]  J. Doudna,et al.  CRISPR-Cas9 Structures and Mechanisms. , 2017, Annual review of biophysics.

[10]  Ira W. Deveson,et al.  Universal alternative splicing of noncoding exons , 2017, bioRxiv.

[11]  Howard Y. Chang,et al.  NONCODING RNA: CRISPRi‐based genome‐scale identification of functional long noncoding RNA loci in human cells , 2017 .

[12]  E. Lander,et al.  Local regulation of gene expression by lncRNA promoters, transcription and splicing , 2016, Nature.

[13]  Jens-Peter Volkmer,et al.  CD47 blocking antibodies restore phagocytosis and prevent atherosclerosis , 2016, Nature.

[14]  Stefan Rose-John,et al.  IL-6 pathway in the liver: From physiopathology to therapy. , 2016, Journal of hepatology.

[15]  R. Hardison,et al.  Unlinking an lncRNA from Its Associated cis Element. , 2016, Molecular cell.

[16]  Max A. Horlbeck,et al.  Nucleosomes impede Cas9 access to DNA in vivo and in vitro , 2016, eLife.

[17]  Mazhar Adli,et al.  Cas9-chromatin binding information enables more accurate CRISPR off-target prediction , 2015, Nucleic acids research.

[18]  Sebastian M. Armasu,et al.  A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease , 2015, Nature Genetics.

[19]  Clifford A. Meyer,et al.  Sequence determinants of improved CRISPR sgRNA design , 2015, Genome research.

[20]  Charles E. Vejnar,et al.  CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo , 2015, Nature Methods.

[21]  Zheng Wei,et al.  CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation , 2015, Bioinform..

[22]  J. L. Mateo,et al.  CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool , 2015, PloS one.

[23]  Ron Weiss,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2014, Nature Methods.

[24]  Toshio Tanaka,et al.  IL-6 in inflammation, immunity, and disease. , 2014, Cold Spring Harbor perspectives in biology.

[25]  Henriette O'Geen,et al.  A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture , 2014, bioRxiv.

[26]  Gang Bao,et al.  CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences , 2014, Nucleic acids research.

[27]  K. Morris,et al.  The rise of regulatory RNA , 2014, Nature Reviews Genetics.

[28]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.

[29]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

[30]  D. Bartel,et al.  lincRNAs: Genomics, Evolution, and Mechanisms , 2013, Cell.

[31]  J. Danesh,et al.  Large-scale association analysis identifies new risk loci for coronary artery disease , 2012, Nature Genetics.

[32]  J. Scheller,et al.  The pro- and anti-inflammatory properties of the cytokine interleukin-6. , 2011, Biochimica et biophysica acta.

[33]  D. Baltimore,et al.  The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules , 2009, Nature Immunology.

[34]  J. Gern.  The Sequence of the Human Genome , 2001, Science.

[35]  D. Mccormick.  Sequence the Human Genome , 1986, Bio/Technology.

[36]  Max A. Horlbeck,et al.  Nucleosomes Impede Cas 9 Access to DNA in vivo and in vitro , 2016 .