Quantification of randomized programmable CRISPR-based toggle switches in Escherichia coli

Recent developments and advances in CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and CIRSPR-associated proteins) systems have ushered a new generation of powerful genetic engineering tools in synthetic biology. In particular, a catalytically ‘dead’ version of CRISPR-Cas proteins that lack nuclease activity can essentially function as a logic NOR gate by selectively binding to a promoter sequence and preventing initiation of transcription by RNA polymerase. In this work, we create programmable and compact genetic toggle switches using pairs of mutually repressible orthogonal CRISPR-based NOR gates and measure the strength of these toggle switches in parallel using a next-generation sequencing method called “Cross-Seq”. Pairs of tandem positioned CRISPR toggle switch with randomized target barcode are inserted into plasmid and transformed into E. coli cells. Each toggle switch output controls a selectable or counter-selectable marker and, by the positively and negatively selecting for bacteria survival, we are able to measure the output of co-repressing NOR gates in all possible combinations and simultaneously quantify the relative strength and stability of dozens of toggle switches in parallel.