These weak interactions in part observe a sequence and structure RNA motif modeled on the Cas9 gRNA. Here we show that Cas9 reproducibly binds to hundreds of human RNAs. We hypothesized that Cas9 might also bind to endogenous eukaryotic RNA transcripts in a CRISPR RNA/gRNA-independent fashion, and that such interactions would be pervasive and potentially consequential in the far more complex transcriptome environment of human cells. It has been shown that Cas9 can associate with transcriptome-wide RNAs in bacteria, although such interactions were attributed to CRISPR RNA-mediated binding 5. The CRISPR-Cas9 system with its programmable synthetic guide RNA (gRNA) 3 has developed into a powerful genome engineering and therapeutic tool 4. Acquired foreign nucleic acids are stored in genomic memory as part of CRISPR arrays, which are then processed into CRISPR RNAs to which Cas proteins bind for downstream foreign nucleic acid recognition and destruction 2. 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.ĬRISPR-Cas (clustered regularly interspaced short-palindromic repeats and CRISPR-associated proteins) systems have evolved in bacteria and archaea as adaptative immune systems defending against phage invaders 1. 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. 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. This association can be partially explained by a model built on gRNA secondary structure and sequence. 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. CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells.
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