Li Yangyang, Li Cen, Fu Yishan, Zhang Quan, Ma Jianing, Zhou Jingwen, Li Jianghua, Du Guocheng, Liu Song
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, 214122, China.
Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
Synth Syst Biotechnol. 2024 Feb 10;9(2):209-216. doi: 10.1016/j.synbio.2024.01.014. eCollection 2024 Jun.
is a highly versatile fungal strain utilized in industrial production. The expression levels of recombinant genes in can be enhanced by increasing the copy number. Nevertheless, given the prolonged gene editing cycle of , a "one-step" strategy facilitating the simultaneous integration of recombinant genes into multiple genomic loci would provide a definitive advantage. In our previous study, a visual multigene editing system (VMS) was designed to knock out five genes, employing a tRNA-sgRNA array that includes the pigment gene and the target genes. Building upon this system, hybrid donor DNAs (dDNAs) were introduced to establish a clustered regularly interspaced short palindromic repeats (CRISPR)-based multiplex integration toolkit. Firstly, a CRISPR-Cas9 homology-directed repair (CRISPR-HDR) system was constructed in by co-transforming the CRISPR-Cas9 plasmid (with a highly efficient sgRNA) and the dDNA, resulting in precise integration of recombinant xylanase gene into the target loci (the β-glucosidase gene , the amylase gene , and the acid amylase gene ). Subsequently, the length of homology arms in the dDNA was optimized to achieve 100% editing efficiency at each of the three gene loci. To achieve efficient multiplex integration in , the CRISPR plasmid pLM2 carrying a sgRNA-tRNA array was employed for concurrent double-strand breaks at multiple loci (, , , and ). Hybrid dDNAs were then employed for repair, including dDNA1-3 (containing expression cassettes without selection markers) and dDNA (for knockout). Among the obtained white colonies (RLM2'), 23.5% exhibited concurrent replacement of the , , and genes with (three copies). Notably, the xynA activity obtained by simultaneous insertion into three loci was 48.6% higher compared to that obtained by insertion into only the locus. Furthermore, this multiple integration toolkit successfully enhanced the expression of endogenous pectinase pelA and lipase CALB. Hence, the combined application of VMS and the CRISPR-HDR system enabled the simultaneous application of multiple selection markers, facilitating the rapid generation in the cell factories.
是一种在工业生产中广泛应用的多功能真菌菌株。通过增加拷贝数可以提高重组基因在其中的表达水平。然而，鉴于其较长的基因编辑周期，一种促进重组基因同时整合到多个基因组位点的“一步法”策略将具有明显优势。在我们之前的研究中，设计了一种可视化多基因编辑系统（VMS）来敲除五个基因，采用了包含色素基因和靶基因的tRNA-sgRNA阵列。在此系统基础上，引入杂交供体DNA（dDNA）以建立基于成簇规律间隔短回文重复序列（CRISPR）的多重整合工具包。首先，通过共转化CRISPR-Cas9质粒（带有高效sgRNA）和dDNA，在中构建了CRISPR-Cas9同源定向修复（CRISPR-HDR）系统，从而使重组木聚糖酶基因精确整合到靶位点（β-葡萄糖苷酶基因、淀粉酶基因和酸性淀粉酶基因）。随后，优化了dDNA中的同源臂长度，以在三个基因位点中的每一个位点实现100%的编辑效率。为了在中实现高效的多重整合，使用携带sgRNA-tRNA阵列的CRISPR质粒pLM2在多个位点（、、、和）同时产生双链断裂。然后使用杂交dDNA进行修复，包括dDNA1-3（包含无选择标记的表达盒）和dDNA（用于敲除）。在获得的白色菌落（RLM2'）中，23.5%表现出、和基因同时被（三个拷贝）取代。值得注意的是，通过同时插入三个位点获得的木聚糖酶A（xynA）活性比仅插入位点获得的活性高48.6%。此外，这个多重整合工具包成功提高了内源性果胶酶pelA和脂肪酶CALB的表达。因此，VMS和CRISPR-HDR系统的联合应用使得能够同时应用多个选择标记，便于在细胞工厂中快速产生。
