Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.
Keywords: CRISPR; CRISPR array synthesis; CRISPR-Cas12a; Cas12a; Gene editing; Genome engineering; Multiplexed; Orthogonal genome engineering; Transcriptional regulation.