Poster abstracts

Poster number 61 submitted by Henry Arthur

Engineering circular guide RNAs for Type I CRISPR mediated gene editing in human cells

Henry C Arthur (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI), Mason T Myers (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI), Renke Tan (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI), Yan Zhang (Department of Biological Chemistry, University of Michigan, Ann Arbor, MI)

Abstract:
Type I CRISPR-Cas is the most abundant phage defense system in nature. It operates in an RNA-guided fashion, using a multi-subunit DNA binding complex called Cascade and a helicase-nuclease Cas3 to shred invading genomes.[1] In 2019, we pioneered the use of Type I CRISPR for Kb-to-Mb sized deletions in the human genome.[2] The Type I CRISPR toolkit has since expanded to include transcriptional activation, DNA integration, and base editing tools, all of which rely on guide RNAs encoded by a plasmid or in chemically modified linear form. These new technologies are promising, but the lack of exploration into circular guide RNAs (circRNA) for Type I gene editing may be hindering their clinical translation. circRNA have two attractive properties compared to linear RNAs. First, they illicit a much lower immune response in human cells due to their lack of phosphorylated ends.[3] Second, they have a longer half-life in cells which may promote higher gene editing efficiency. Here we develop a robust strategy using self-cleaving ribozymes to produce in vitro circRNA for the Type I-C CRISPR-Cas3 gene editor. We electroporated these circRNA along with mRNAs encoding Nla I-C casproteins into HEK293T-GFP reporter cells and found that our circRNA enabled 80% disruption editing of GFP, putting it on par with chemically modified linear guides. We also determined that the method of circRNA purification had significant effects on our editing outcome. Our results set the stage for future efforts to advance Type I CRISPR into the clinic using circRNAs.

References:
1. Liu, T.Y. & Doudna, J.A. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation
2. Dolan & Hou et al., Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas
3. Wesselhoeft et al., RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo

Keywords: circular RNA, Ribozyme, CRISPR gene editing