sb5b00147_si_002.zip (72.13 kB)
Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9
dataset
posted on 2016-01-15, 00:00 authored by Andriy Didovyk, Bartłomiej Borek, Jeff Hasty, Lev TsimringThe progress in development of synthetic
gene circuits has been
hindered by the limited repertoire of available transcription factors.
Recently, it has been greatly expanded using the CRISPR/Cas9 system.
However, this system is limited by its imperfect DNA sequence specificity,
leading to potential crosstalk with host genome or circuit components.
Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent,
affecting the modularity of Cas9-based transcription factors. In this
paper we address the problems of specificity and modularity by developing
a computational approach for selecting Cas9/gRNA transcription factor/promoter
pairs that are maximally orthogonal to each other as well as to the
host genome and synthetic circuit components. We validate the method
by designing and experimentally testing four orthogonal promoter/repressor
pairs in the context of a strong promoter PL from phage
lambda. We demonstrate that these promoters can be interfaced by constructing
double and triple inverter circuits. To address the problem of modularity
we propose and experimentally validate a scheme to predictably incorporate
orthogonal CRISPR/Cas9 regulation into a large class of natural promoters.