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• W4214922503 abstract "Abstract 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 one single replicative vector, for the model cyanobacteria 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 overall performed better, reaching e.g. 100% for insertion of a FLAG-tag onto rbcL . Importantly, the single-vector CRISPR/Cas9 system described herein was also shown to mediate 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, adding to the usefulness of this system. Further, a vector curing system that is separately induced by nickel and contained on the CRISPR/Cas9 vector itself, improved curing efficiencies by roughly 4-fold, enabling the final mutants to become truly markerless." @default.
• W4214922503 created "2022-03-05" @default.
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• W4214922503 date "2022-03-02" @default.
• W4214922503 modified "2023-10-15" @default.
• W4214922503 title "Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in <i>Synechocystis</i> sp. PCC 6803" @default.
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• W4214922503 doi "https://doi.org/10.1101/2022.03.02.482598" @default.
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