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Small-molecule inhibitors of CRISPR/Cas13 enable versatile control of RNA knockdown in mammalian cells

ABSTRACT: Abstract: CRISPR/Cas13 has emerged as a leading platform for carrying out RNA knockdown and editing experiments, and presents an attractive therapeutic alternative to DNA editing technologies. However, strategies to directly modulate its activity in a fast, reliable, and generalizable manner are lacking. Here, we carry out a high- throughput screen to identify small-molecule inhibitors of LwaCas13a in order to enable titration of RNA knockdown and spatiotemporal control. We identify 6 lead hits that potently inhibit on-target and collateral Cas13 RNA cleavage but not pre-crRNA processing activity. We show that these compounds act via non-competitive and mixed mechanisms, and are selective for a subset of Cas13 homologs. Moreover, we demonstrate that two compounds, CID5483251 and HTS10308, inhibit RNA knockdown by exogenous Cas13 in mammalian cells at subtoxic doses, and can be used to tune mRNA and corresponding protein levels in a dose- and time-dependent manner. This study delivers new tools for achieving versatile control of Cas13 activity in mammalian cells, and establishes a foundation for the design of additional Cas13 inhibitors.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Venita Sitahal

LAB HEAD: Derek Wilson

PROVIDER: PXD063296 | Pride | 2026-07-17

REPOSITORIES: Pride

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			Action	DRS
	240618_LwaCas13a_0sec_01_vial01_IA_Log.txt	Txt
	240618_LwaCas13a_0sec_01_vial01_IA_final_fragment.csv	Csv
	240618_LwaCas13a_0sec_01_vial01_IA_final_peptide.csv	Csv
	240618_LwaCas13a_0sec_01_vial01_IA_final_protein.csv	Csv
	240618_LwaCas13a_0sec_01_vial01_IA_workflow.bin	Other

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