Members of the genus Clostridium represent a diverse
assemblage of species exhibiting both medical and industrial importance.
Deriving both a greater understanding of their biology, while at the
same time enhancing their exploitable properties, requires effective
genome editing tools. Here, we demonstrate the first implementation
in the genus of theophylline-dependent, synthetic riboswitches exhibiting
a full set of dynamic ranges, also suitable for applications where
tight control of gene expression is required. Their utility was highlighted
by generating a novel riboswitch-based editing toolRiboCasthat
overcomes the main obstacles associated with CRISPR/Cas9 systems,
including low transformation efficiencies and excessive Cas9 toxicity.
The universal nature of the tool was established by obtaining chromosomal
modifications in C. pasteurianum, C. difficile, and C. sporogenes, as well as by carrying
out the first reported example of CRISPR-targeted gene disruption
in C. botulinum. The high efficiency (100% mutant
generation) and ease of application of RiboCas make it suitable for
use in a diverse range of microorganisms.
Establishing various synthetic biology tools is crucial
for the
development of cyanobacteria for biotechnology use, especially tools
that allow for precise and markerless genome editing in a time-efficient
manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system,
contained on a single replicative vector, for the model cyanobacterium
Synechocystis
sp. PCC 6803. A theophylline-responsive
riboswitch allowed tight control of Cas9 expression, which enabled
reliable transformation of the CRISPR/Cas9 vector into
Synechocystis
. Induction of the CRISPR/Cas9 mediated
various types of genomic edits, specifically deletions and insertions
of varying size. The editing efficiency varied depending on the target
and intended edit; smaller edits performed better, reaching, e.g.,
100% for insertion of a FLAG-tag onto
rbcL
. Importantly,
the single-vector CRISPR/Cas9 system mediated multiplexed editing
of up to three targets in parallel in
Synechocystis
. All single-target and several double-target mutants were also fully
segregated after the first round of induction. Lastly, a vector curing
system based on the nickel-inducible expression of the toxic
mazF
(from
Escherichia coli
) was added to the CRISPR/Cas9 vector. This inducible system allowed
for curing of the vector in 25–75% of screened colonies, enabling
edited mutants to become markerless.
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