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- W4386863344 abstract "ABSTRACT DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Tr anscription- a ssociated C as9 T argeting (TraCT). Engineered TraCT is enabled when suboptimal PAM interactions limit basal activity in vivo and when one or more nascent RNA substrates are still tethered to the actively transcribing target DNA in cis . We further show that this phenomenon can be exploited for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9’s targeting activity at specific DNA sites may be engineered without modifying Cas9’s core domains and guide RNA components or their expression levels. More broadly, it establishes RNA binding in cis as a mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotes." @default.
- W4386863344 created "2023-09-20" @default.
- W4386863344 creator A5012078465 @default.
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- W4386863344 creator A5065792746 @default.
- W4386863344 creator A5075536007 @default.
- W4386863344 creator A5089509256 @default.
- W4386863344 date "2023-09-18" @default.
- W4386863344 modified "2023-10-15" @default.
- W4386863344 title "Engineered transcription-associated Cas9 targeting in eukaryotic cells" @default.
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