Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging
Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging
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DOI：
10.1093/genetics/iyab180
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发表时间：
2021-10-23
期刊：
影响因子：
3.3
通讯作者：
Madhani, Hiten D.
中科院分区：
文献类型：
--
作者：
Huang, Manning Y.;Joshi, Meenakshi B.;Madhani, Hiten D.
新型隐球菌是真菌性脑膜炎最常见的病因，是一种具有完整性周期的担子菌单倍体芽殖酵母。利用生物转化和长同源臂对基因组进行同源重组修饰是可行的，但该方法难度大、不可靠。最近，多个研究小组已经报道了使用CRISPR-Cas9作为生物学的替代方法，但仍然需要长同源臂，限制了该方法的实用性。由于先前研究中使用的化脓性链球菌Cas9衍生物并未优化在新生C. formans中的表达，因此我们设计、合成并测试了一个完全优化的新生C. formans (Cno) Cas9。我们发现，在广泛使用的KN99 α C. neoformans菌株中，仅含有常见的C. neoformans密码子和一致的C. neoformans内含子的Cas9与TEF1启动子和终止子以及核定位信号（Cno Cas9或“CnoCAS9”）可靠地实现了基因组编辑。此外，通过将短的（50 bp）同源臂连接到由合成寡核苷酸和PCR扩增产生的标记dna上的供体完成编辑。我们还证明，事先稳定整合CnoCAS9进一步提高了转化效率和同源重组效率；重要的是，这种操作不会影响动物的毒力。我们还实现了一个通用标记模块，包含一个密码子优化的荧光蛋白（mNeonGreen）和一个串联钙调蛋白结合肽- 2x FLAG标签，该标签允许通过短同源定向重组修饰相应基因的蛋白质的定位和纯化研究。这些工具使短同源基因组工程在新生生物。
Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99 alpha C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.