Optimization of Cas12a for multiplexed genome-scale transcriptional activation.

Autor:	Griffith AL; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Zheng F; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., McGee AV; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Miller NW; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Szegletes ZM; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Reint G; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Gademann F; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Nwolah I; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Hegde M; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Liu YV; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Goodale A; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA., Doench JG; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA 02142, USA.
Jazyk:	angličtina
Zdroj:	Cell genomics [Cell Genom] 2023 Sep 01; Vol. 3 (9), pp. 100387. Date of Electronic Publication: 2023 Sep 01 (Print Publication: 2023).
DOI:	10.1016/j.xgen.2023.100387
Abstrakt:	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. Competing Interests: J.G.D. consults for Microsoft Research, Abata Therapeutics, Maze Therapeutics, BioNTech, Sangamo, and Pfizer. J.G.D. consults for and has equity in Tango Therapeutics. J.G.D. serves as a paid scientific advisor to the Laboratory for Genomics Research, funded in part by GSK, and the Innovative Genomics Institute, funded in part by Apple Tree Partners. J.G.D. receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, Merck, and Vir Biotechnology. J.G.D.’s interests are reviewed and managed by the Broad Institute in accordance with its conflict-of-interest policies. (© 2023 The Author(s).)
Databáze:	MEDLINE
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