Scholarlink

<p>Single-cell perturbation (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a’s guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery during single-cell RNA sequencing library preparation. To overcome the self-processing constraint, we optimized pre-crRNA expression vectors and established a degron-based, enhanced Cas12a system for gene knock-out. As demonstrated across cell types, target genes, and with a minimized guide RNA library, this platform allows for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome in single cells. Additionally, we show that HyperLbCas12a outperforms other existing variants for multiplexed gene suppression. While the rapid reversibility of this repressor highlights specific kinetic constraints for degron-based single-cell recording, the system provides a potent, modular tool for contexts requiring tunable, transient silencing. Together, this suite of technologies greatly expands the possibilities for future Perturb-seq efforts and broader application of Cas12a for genetic disruption at scale.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

A tunable Cas12a platform for single-cell perturbation screening and CRISPRi

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年6月2日

查看全文
  • Valentina Snetkova,
  • Carolina Galan,
  • Romain Lopez,
  • Antonio R. Rios,
  • Takamasa Kudo,
  • Kristel Dorighi,
  • Søren Warming,
  • Benjamin J. Haley

摘要

Single-cell perturbation (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a’s guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery during single-cell RNA sequencing library preparation. To overcome the self-processing constraint, we optimized pre-crRNA expression vectors and established a degron-based, enhanced Cas12a system for gene knock-out. As demonstrated across cell types, target genes, and with a minimized guide RNA library, this platform allows for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome in single cells. Additionally, we show that HyperLbCas12a outperforms other existing variants for multiplexed gene suppression. While the rapid reversibility of this repressor highlights specific kinetic constraints for degron-based single-cell recording, the system provides a potent, modular tool for contexts requiring tunable, transient silencing. Together, this suite of technologies greatly expands the possibilities for future Perturb-seq efforts and broader application of Cas12a for genetic disruption at scale.