tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9

Ankush Auradkar, Annabel Guichard, Saluja Kaduwal, Marketta Sneider and Ethan Bier ()
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Ankush Auradkar: University of California, San Diego
Annabel Guichard: University of California, San Diego
Saluja Kaduwal: University of California, San Diego
Marketta Sneider: University of California, San Diego
Ethan Bier: University of California, San Diego

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14–15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.

Date: 2023
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DOI: 10.1038/s41467-023-40836-3

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