Journal article
Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision
C Shembrey, R Yang, J Casan, W Hu, H Chen, GJ Singh, T Sadras, K Prasad, J Shortt, RW Johnstone, JA Trapani, PG Ekert, M Fareh
Science Advances | Published : 2024
Abstract
Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRA..
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Grants
Awarded by Gilead Research Scholars
Funding Acknowledgements
This work was supported by a Cancer Council Victoria Ventures grant (829606 to M.F., P.G.E., and J.A.T.), MAP MRNA Victoria grant (RCH0153742 to M.F.), and a Peter MacCallum Cancer Centre strategic plan funding in partnership with the Children's Cancer Institute Australia (to M.F.). C.S. is supported by an Australia and New Zealand Sarcoma Association (ANZSA) Research Grant. T.S. is supported by a Gilead Research Scholars Award and funding from The Kid's Cancer Project Mid- Career Fellowship. P.G.E. acknowledges the funding support of the Luminesce Alliance for "Functional Genomics Platform, Translational Tumor Biology." R.W.J. acknowledges funding from the Cancer Council Victoria and The Kids Cancer Project. The authors received additional funding from the National Health and Medical Research Council Ideas Grant 2028235 (to P.G.E.) and Investigator Grant APP2016820 (to R.W.J.). We also acknowledge the support of Perpetual Trustees and the Samuel Nissen Foundation.