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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Single-cell perturbation (Perturb-seq) screens commonly use Cas9 for gene knock-out.
• Cas12a offers advantages for multiplexed guide RNA expression but is underexplored due to self-processing issues and recovery challenges in single-cell RNA sequencing.
• Existing Cas12a systems face limitations in pre-crRNA recovery and gene editing efficiency.

Purpose of the Study:

• To overcome Cas12a's pre-crRNA self-processing constraint for improved Perturb-seq applications.
• To develop and validate a degron-based, enhanced Cas12a system for efficient gene knock-out and transcriptome analysis in single cells.
• To evaluate the performance of Cas12a variants for multiplexed gene suppression and transient gene silencing.

Main Methods:

• Optimization of pre-crRNA expression vectors for Cas12a.
• Development of a degron-based, enhanced Cas12a system for gene knock-out.
• Application of the system across diverse cell types and target genes with a minimized guide RNA library.
• Assessment of pre-crRNA detection and gene editing-induced transcriptomic effects in single cells.
• Comparative analysis of Cas12a variants, including HyperLbCas12a, for multiplexed gene suppression.

Main Results:

• The optimized Cas12a system accurately detects pre-crRNAs and gene editing effects on the transcriptome in single cells.
• The degron-based system enables efficient gene knock-out across various cell types and targets.
• HyperLbCas12a demonstrated superior performance for multiplexed gene suppression compared to other variants.
• The system allows for tunable, transient gene silencing, highlighting kinetic constraints for degron-based recording.

Conclusions:

• The developed Cas12a platform overcomes self-processing limitations, enabling robust single-cell genetic disruption.
• This technology significantly expands the utility of Cas12a for large-scale Perturb-seq and genetic screening.
• The findings provide a potent and modular tool for researchers studying gene function and regulation at the single-cell level.