Forrer Selina, Arentshorst Mark, Koolth Valappil Prajeesh, Visser Jaap, Ram Arthur F J
Institute Biology Leiden, Microbial Sciences, Fungal Genetics and Biotechnology, Leiden University, Sylviusweg 72, Leiden, 2333 BE, The Netherlands.
Fungal Biol Biotechnol. 2024 Oct 15;11(1):15. doi: 10.1186/s40694-024-00184-3.
Aspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.
Here we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13-20% of the transformants while 80-87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35-61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85-90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.
The awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.
黑曲霉以其高蛋白分泌能力而闻名，因此是同源和异源蛋白生产的重要细胞工厂。使用强启动子和多个基因拷贝是增加目标基因（GOI）基因表达和蛋白产量的常用策略。我们最近提出了一种两步CRISPR/Cas9介导的方法，其中在基因组的预定位点引入糖化酶（glaA）着陆位点（GLS）（步骤1），随后用GOI的拷贝填充这些位点（步骤2）以实现GOI的高表达。
在这里我们表明，在ku70缺陷型黑曲霉菌株（Δku70）中，从而排除非同源末端连接（NHEJ）作为修复双链DNA断裂（DSB）的机制，染色体glaA位点或同源GLS可用于修复Cas9诱导的DSB，从而与含有GOI的供体DNA的整合竞争。在没有外源添加供体DNA的情况下，DSB用位于其他染色体上的同源染色体DNA修复（染色体间修复），或者更高效地用位于同一条染色体上的同源DNA片段修复（染色体内修复）。发现单拷贝染色体间同源性DNA修复发生在13%-20%的转化体中，而80%-87%的转化体由外源添加的供体DNA修复。染色体修复的效率取决于基因组中潜在供体DNA序列的拷贝数。五个同源DNA序列的存在导致由染色体DNA修复的转化体数量增加（35%-61%）。发现在没有供体DNA的情况下，基于染色体内同源性的DSB修复效率比染色体间修复更受青睐（85%-90%）。还发现染色体内修复也是存在供体DNA时DNA修复的首选方式，并且发现其与位点有关。
同源染色体DNA修复可以与供体DNA竞争修复DSB，从而影响使用CRISPR/Cas9介导的基因组编辑构建多拷贝菌株的效率，这一认识是工业菌株设计中需要考虑的重要因素。
