Pulling the genome in opposite directions to dissect gene networks

Abstract

Orthogonal CRISPR-Cas systems have been integrated into combinatorial screens to decipher complex genetic relationships in two recent studies.

Fig. 1

Boettcher et al. [3] and Najm et al. [4] demonstrate combinatorial bi-directional CRISPR screens integrating gene activation and gene knockout platforms. a The dual guide RNA (gRNA) expression cassettes are synthesized on arrays with pools of gRNAs compatible with SpydCas9 and SauCas9 that target a distinct set of gene promoters and gene coding sequences, respectively. b Each cell is engineered to express both a SpydCas9 activator and the SauCas9 nuclease, and also receives a single dual gRNA cassette, leading to activation and knockout of a unique gene pair. The pool of cells with diverse gRNA pairs is selected based on unique phenotypes conferred by these divergent gene perturbations, which are identified by sequencing the gRNA cassettes. c Various combinations of orthogonal Cas9 effectors enable concurrent control of transcriptional activation, repression, knockouts, base editing, epigenome alteration, and/or imaging