- Book Chapter
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. Show more
Book titleMammalian Cell Engineering. Methods and Protocols
Journal / seriesMethods in Molecular Biology
Pages / Article No.
SubjectGenome engineering; CRISPR; Cas12a; CRISPR-Cas12a; Multiplexed; Orthogonal genome engineering; CRISPR array synthesis; Gene editing; Transcriptional regulation
Organisational unit09580 - Platt, Randall / Platt, Randall
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