Nalley Matthew J, Banerjee Sanjita, Huang Manning Y, Madhani Hiten D
Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th St., San Francisco, CA 94158, USA.
G3 (Bethesda). 2025 Jun 3. doi: 10.1093/g3journal/jkaf118.
We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with two separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires two manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology-dependent genome editing after selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.
我们最近描述了基于CRISPR/Cas9的短同源依赖性基因组工程，该工程应用于人类真菌病原体新型隐球菌，它是一种单倍体出芽酵母，是真菌性脑膜炎最常见的病因，也是一种新兴的模式生物。这是通过将稳定表达密码子优化的Cas9的菌株与两个单独的DNA分子进行电穿孔来实现的，一个DNA分子编码两侧带有短同源臂的选择标记，另一个DNA分子在U6 snRNA启动子的控制下编码sgRNA。然而，相对于不期望的非同源末端连接（NHEJ）事件，所需的同源依赖性修复效率可能较低且存在差异。在这里，我们描述了在新型隐球菌中实现极其高效（约99%）的同源依赖性基因组编辑的方法和菌株。这种高效方法需要两个操作步骤。首先，我们将表达sgRNA的片段放入两侧带有靶向同源序列的含标记DNA中；因此，只需将单个DNA分子导入细胞。其次，我们使用了非同源末端连接因子Ku80（由YKU80编码）的突变菌株。我们还报告了yku80::amdS突变菌株的构建，该菌株含有一个插入突变，可通过直接重复序列之间的重组无疤痕地去除。这使得在使用氟乙酰胺针对amdS标记进行选择后，同源依赖性基因组编辑后YKU80能够实现功能恢复。这种方法最大限度地减少了在下游实验中使用Ku缺陷菌株所记录的缺点。最后，我们描述了一个质粒系列，该系列能够快速克隆用于新型隐球菌基因组操作的sgRNA-标记构建体，包括基因缺失和C末端标记。这些方法、菌株和质粒加速了新型隐球菌的基因组操作。
