Tuning programmable CRISPR-based toggle switches with buffer sites in Escherichia coli

Recent developments and advances in CRISPR-Cas systems have ushered a new generation of powerful genetic engineering tools in synthetic biology. In particular, a catalytically ‘dead’ version of Cas proteins that lack nuclease activity can essentially function as a logic NOT gate by selectively binding to a promoter and preventing transcription initiation. In this work, we first create programmable genetic toggle switches(TSs) using pairs of mutually repressible orthogonal CRISPR-based NOT gates and measure the strength of these TSs using massive parallel CRISPRi assay called “Xseq”. Specifically, hundred pairs of CRISPR nodes with different barcoded targets are simultaneously cloned into a plasmid that contains a TetA-SacB cassette as a reporter, then transformed into E. coli. Each cell has a single TS construct. By selecting cell survival under sucrose and tetracycline conditions, we are able to sort out matched CRISPR TSs among numerous randomized promoter-target pairs and quantify relative strength of each NOT gate in all possible combination at the same time. Later, adding a second plasmid that shares the same target as the TS and competes with TS for dCas pool, we can tune the efficiency of each NOT gate via manipulating the number of buffers, and thus the performance of TS.