Crosstalk between CRISPR-Cas9 and the human transcriptome

Aaron A Smargon,Brian A Yee,Assael A Madrigal,Gene W Yeo,Jasmine R Mueller,Kevin D Dong

doi:10.1038/s41467-022-28719-5

Abstract

CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.

Highlights

CRISPR-Cas[9] expression independent of its cognate synthetic guide RNA causes widespread genomic DNA damage in human cells
Under guide RNA (gRNA) co-expression such human RNA interactions do not correlate with Cas[9] genomic DNA binding or cleavage, they do correlate with elevated RNA editing
To test our central hypothesis, we performed enhanced cross-linking and immunoprecipitation followed by sequencing with anti-V5 and anti-FLAG antibodies in transfected human HEK 293T cells (Fig. 1a), as has been conducted for human RNA-binding proteins (RBPs) with ineffective antibodies[6]

Summary

Introduction

CRISPR-Cas[9] expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas[9] can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas[9] in human cells and find that Cas[9] reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Implications of this study for the use of CRISPR-Cas systems in human cells include off-target RNA editing or modification and bound transcript RNA/protein level changes

Methods

Results

Conclusion