PRJNA530879
Massively multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts
The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we developed a CRISPR-Cas12a-based platform to enable constitutive, conditional, inducible, orthogonal and massively multiplexed genome engineering using a single transcript. We encode both the CRISPR array and Cas12a in a single mRNA and improve the performance of this system by adding a stabilizer tertiary RNA structure. This platform, termed single-transcript Cas12a (SiT-Cas12a), when coupled with conditional or inducible expression systems enables tunable production of both Cas12a and CRISPR RNA (crRNA) – facilitating spatial and temporal control of genome engineering. In addition, through optimization of SiT-Cas12a transcriptional repressor and activator effector fusion protein architectures, we achieve greater efficiencies compared to previous Cas12a-based technologies and also demonstrate massively multiplexed transcriptional control. Finally, by truncating the spacer sequence within the crRNA, our system enables orthogonal gene editing and transcriptional regulation. SiT-Cas12a provides a powerful platform for enabling massively multiplexed genome engineering applications and, in the future, may be used to interrogate and orchestrate the sophisticated genetic programs underlying complex cell behaviours.