CRISPR/Cas9-mediated genome editing of <i>Shewanella oneidensis</i> MR-1 using a broad host-range pBBR1-based plasmid

Abstract

Commonly used CRISPR/Cas9 systems such as the type-II system from Streptococcus pyogenes are composed of an RNA-guided endonuclease (Cas9) and a single-guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) complementary to a 20-nt target DNA sequence as well as a transactivating crRNA (tracrRNA) that recruits the crRNA to Cas9 (Doudna and Charpentier, 2014). The 20-nt target DNA sequence is followed by a 5¢-NGG-3¢ protospacer adjacent motif (PAM) (Choudhary et al., 2015; Nayak and Metcalf, 2017). The Cas9 complex, guided by the sgRNA, cleaves the target sequence approximately three base pairs upstream of the PAM to generate a double-strand DNA break (DSB), which triggers mutations through nonhomologous end joining (NHEJ) repair or homologydirected repair (HDR) (Cho et al., 2018; Ran et al., 2013). Despite extensive use of genome-editing technologies in eukaryotes, their use in bacteria is limited because of the lack of vectors for CRISPR/Cas9 systems. Several cas9-expressing plasmids applicable to genome editing of bacteria are available (

Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria

“…Of these, the pBBR1 plasmid is small, has a broad range, relatively high stability, and is maintained at a medium copy number. Specifically, BBR1 and derivatives have been shown to replicate in all major divisions of Pseudomonadota , and as of this writing, at least 33 species (Figure A). − In pBBR1, the Rep protein is required for replication, and in all known cases expression is controlled by DNA elements (i.e., promoter and RBS) native to the pBBR1 plasmid (Figure B). Thus, a backbone with a replicon based on BBR1 and compatible with the CIDAR Modular Cloning standard could extend well-known synthetic biology parts across a wide range of bacterial hosts (Figure A).…”

Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production

“…Using this system to mutant the crp gene encoding a transcriptional regulator of the CRP/FNR family (cyclic monophosphate receptor protein) can effectively mediate double-stranded DNA breaks and repair. 96 However, the CRISPR/Cas genome editing is susceptible to off-targeting, resulting in lower editing efficiency and accuracy. Wu et al thus developed a novel CRISPR/Cas genome editing system combined with recE/recT recombinase in Shewanella algae , enabling the precise deletion or repair of the dsDNA breaks (DSBs) generated by CRISPR/Cas through homologous recombination (HR) with DNA templates.…”

“…2.2.2 Homologous recombination. Modifying target genes in EAMs using the Cre-lox system, 92,93 in-frame deletion, 94,95 and gene editing techniques, 96 have been conducive for studying the functional relationships between genes and electroactivity. The Cre-lox system, first developed by Marx et al in Gram-negative bacteria, comprised a dual plasmid system, which included an allele exchange vector bearing a kanamycin cassette with two loxP sites, one at each end and the IncP plasmid-making Cre recombinase.…”

Application of synthetic biology in living functional materials

“…2014年, Lu实验 室 [30] , 开关、振荡器 等被开发并成功地运行于不同的模式生物, 丰富了细 胞编辑的能力. 同时, 随着CRISPR [44] 的发展及普及、 基因合成成本的降低 [45] 、生物编辑的成本及难度大幅 下降 [46] 等, 这些基因编辑工具都为编辑活体功能材料 The fast development of synthetic biology has greatly promoted the understanding of biology and broadened the application of engineered biological systems. Numerous cell-based circuits are assembled using modular biological building blocks.…”