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<p>The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR–Cas13. We show that collateral activity only occurs with high <i>Rfx</i>Cas13d expression. Using low-copy <i>Rfx</i>Cas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.</p>

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Precise RNA targeting with CRISPR–Cas13d

  • 期刊论文

  • 发表日期：

    2025年2月11日

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  • Sydney K. Hart,
  • Simon Müller,
  • Hans-Hermann Wessels,
  • Alejandro Méndez-Mancilla,
  • Gediminas Drabavicius,
  • Olivia Choi,
  • Neville E. Sanjana

摘要

The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR–Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.