Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10⁻⁶ transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.

Synechococcus elongatus UTEX 2973 是测得的最快的蓝藻倍增时间之一，使其成为代谢工程的重要候选者。然而，由于蓝藻的寡倍体或多倍体性质以及对独特抗生素抗性标记的依赖，依赖同源重组的传统基因工程方法效率低下、劳动密集型和耗时。CRISPR-Cas9 已成为多种生物中有效且多功能的编辑平台，但其在蓝藻工程中的应用受到Cas9固有毒性的限制导致转化效率低下。在这里，我们证明了单质粒 CRISPR-Cas9 系统 pCRISPOmyces-2 可以有效地敲入来自大肠杆菌的截短硫酯酶基因以产生产生游离脂肪酸 (FFA) 的Syn 2973 突变体。为此，三个参数评估了与基于 pCRISPOmyces-2 的质粒缀合后产生受体菌落的效果：1) 一种改进的缀合方案，称为条纹缀合，2) 编码 RecJ 外切核酸酶的基因的缺失，和 3) 单向导 RNA (sgRNA) 序列. 使用条纹偶联方案和Syn的 Δ recJ突变株2973，pCRISPomyces-2 质粒的结合效率可以比野生型 (WT) 提高 750 倍，结合效率为 2.0 × 10 ^-6转结合体/受体细胞。虽然仅删除 RecJ 外切核酸酶使缀合效率比 WT 增加了 150 倍，但在具有 Δ recJ背景的 FFA 产生突变体中，FFA 的产生受到损害，并且大量产生 FFA 的分离株表明自发的潜在增加突变率。发现 sgRNA 序列对于实现所需的 CRISPR-Cas9 介导的敲入突变至关重要，因为 sgRNA 会影响共轭效率、同源重组体的可能性以及工程菌株中游离脂肪酸的产生。