Optimization of Cas12a for multiplexed genome- scale transcriptional activation

Cas12a CRISPR technology, unlike Cas9, allows for multiplexing 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 to 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 we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale. Overall design: Bulk RNA sequencing (RNA-seq) on MelJuSo cells expressing 5x-tag-dCas12a-VP64 and nanobody-p65 or nanobody-VP64 with or without the cassette containing three CD4-targeting guides. There are 5 unique samples in total and each is screened in triplicates.