Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

Amita Tiyaboonchai (), Anne Vonada, Jeffrey Posey, Carl Pelz, Leslie Wakefield and Markus Grompe
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Amita Tiyaboonchai: Oregon Health & Science University
Anne Vonada: Oregon Health & Science University
Jeffrey Posey: Oregon Health & Science University
Carl Pelz: Oregon Health & Science University
Leslie Wakefield: Oregon Health & Science University
Markus Grompe: Oregon Health & Science University

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: Abstract Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.

Date: 2022
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DOI: 10.1038/s41467-022-35097-5

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