Crosstalk between CRISPR-Cas9 and the human transcriptome.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 is a powerful gene-editing tool, but its off-target effects, including DNA damage independent of guide RNA (gRNA), are a concern.
• Understanding Cas9's interactions with cellular components beyond its intended DNA target is crucial for refining CRISPR technology.

Purpose of the Study:

• To investigate whether Cas9 can interact with endogenous human RNA transcripts independently of its synthetic guide RNA (gRNA).
• To characterize the nature and implications of Cas9-RNA interactions in human cells.

Main Methods:

• Performed enhanced CLIP (eCLIP) of Cas9 in human cells to identify endogenous RNA interactors.
• Developed a model based on gRNA secondary structure and sequence to explain Cas9-RNA associations.
• Correlated Cas9 binding sites with published data on Cas9 DNA binding, cut sites, and RNA editing.

Main Results:

• Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts in a gRNA-independent manner.
• Cas9-RNA interactions can be partially predicted by gRNA structure and sequence.
• Transcriptome-wide Cas9 binding sites on RNA do not correlate with genome-wide DNA binding or cleavage sites when gRNA is co-expressed.
• Low-affinity Cas9-human RNA interactions, termed CRISPR crosstalk, correlate with elevated transcriptome-wide RNA editing.

Conclusions:

• Human RNAs do not broadly guide Cas9 to bind and cleave genomic DNA.
• Cas9 exhibits inherent RNA-binding capabilities, termed CRISPR crosstalk, which can impact cellular RNA.
• These findings highlight a cellular and RNA-mediated impact intrinsic to CRISPR-Cas systems, independent of DNA-targeting.