Optimization of Cas12a for multiplexed genome-scale transcriptional activation

• Published in: Cell genomics Vol. 3; no. 9; p. 100387
• Main Authors: Griffith, Audrey L., Zheng, Fengyi, McGee, Abby V., Miller, Nathan W., Szegletes, Zsofia M., Reint, Ganna, Gademann, Fabian, Nwolah, Ifunanya, Hegde, Mudra, Liu, Yanjing V., Goodale, Amy, Doench, John G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.09.2023; Elsevier
• Subjects: Cas12a; CRISPR activation; functional genomics; genetic screens; Technology; CRISPR activation; functional genomics; genetic screens; Cas12a
• ISSN: 2666-979X; 2666-979X
• DOI: 10.1016/j.xgen.2023.100387

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results with those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale. [Display omitted] •Development of a nanobody-based recruitment system for Cas12a-based CRISPRa•Genome-wide CRISPRa screens reveal selumetinib-sensitizing and -resistance genes•Design guidance for multiplexed Cas12a CRISPRa Cas12a allows for easy multiplexing of guide RNAs in a single construct. CRISPRa, in which transactivation domains are used to overexpress endogenous genes, has been optimized using Cas9 but not Cas12a. Griffith et al. optimize Cas12a-based CRISPRa and demonstrate that recruitment using the ALFA tag and a corresponding nanobody enables potent gene activation across multiple genes simultaneously. Then, they perform genome-wide Cas12a-based CRISPRa screens, compare results with screens using other technologies, and derive guidelines for library design.