School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm 17121, Sweden.
BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, The University of Nottingham, Nottingham NG7 2RD, U.K.
ACS Synth Biol. 2022 Sep 16;11(9):3100-3113. doi: 10.1021/acssynbio.2c00375. Epub 2022 Aug 15.
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 sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into. 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 . Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel in. 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 (from ) 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.
建立各种合成生物学工具对于开发用于生物技术的蓝藻至关重要，特别是那些能够以高效、精确和无标记的方式进行基因组编辑的工具。在这里，我们描述了一个基于单个复制载体的、针对模式蓝藻 sp. PCC 6803 的诱导型 CRISPR/Cas9 系统。一个茶碱响应型 riboswitch 可以紧密控制 Cas9 的表达，从而实现了 CRISPR/Cas9 载体可靠地转化为。诱导 CRISPR/Cas9 介导了各种类型的基因组编辑，特别是大小不同的缺失和插入。编辑效率取决于靶标和预期的编辑；较小的编辑效果更好，例如，在 插入 FLAG 标签的效率达到 100%。重要的是，该单载体 CRISPR/Cas9 系统能够在 中平行介导多达三个靶标的多路编辑。所有单靶标和几个双靶标突变体在第一轮诱导后也完全分离。最后，我们在 CRISPR/Cas9 载体中添加了基于镍诱导表达的毒性基因 （来自 ）的载体消除系统。这个诱导型系统可以使 25-75%的筛选菌落中消除载体，从而使编辑突变体成为无标记的。
