Abstract：
The nonconventional methylotrophic yeast Komagataella phaffii is widely applied in the production of industrial enzymes, pharmaceutical proteins, and various high-value chemicals. The development of robust and versatile genome editing tools for K. phaffii is crucial for the design of increasingly advanced cell factories. Here, we first developed a base editing method for K. phaffii based on the CRISPR-nCas9 system. We engineered 24 different base editor constructs, using a variety of promoters and cytidine deaminases (CDAs). The optimal base editor (PAOX2*-KpA3A-nCas9-KpUGI-DAS1TT) comprised a truncated AOX2 promoter (PAOX2*), a K. phaffii codon-optimized human APOBEC3A CDA (KpA3A), human codon-optimized nCas9 (D10A), and a K. phaffii codon-optimized uracil glycosylase inhibitor (KpUGI). This optimal base editor efficiently performed C-to-T editing in K. phaffii, with single-, double-, and triple-locus editing efficiencies of up to 96.0%, 65.0%, and 5.0%, respectively, within a 7-nucleotide window from C-18 to C-12. To expand the targetable genomic region, we also replaced nCas9 in the optimal base editor with nSpG and nSpRy, and achieved 50.0%-60.0% C-to-T editing efficiency for NGN-protospacer adjacent motif (PAM) sites and 20.0%-93.2% C-to-T editing efficiency for NRN-PAM sites, respectively. Therefore, these constructed base editors have emerged as powerful tools for gene function research, metabolic engineering, genetic improvement, and functional genomics research in K. phaffii.
摘要：
非常规甲基营养酵母Komagataellaphafii被广泛应用于工业酶的生产中，药用蛋白质,和各种高价值化学品。为K.phafii开发强大而通用的基因组编辑工具对于设计越来越先进的细胞工厂至关重要。这里,我们首先基于CRISPR-nCas9系统开发了一种用于K.phafii的碱基编辑方法。我们设计了24种不同的基础编辑器结构，使用各种启动子和胞苷脱氨酶（CDAs）。最佳碱基编辑器（PAOX2*-KpA3A-nCas9-KpUGI-DAS1TT）包含截短的AOX2启动子（PAOX2*），一种K.phafii密码子优化的人APOBEC3ACDA（KpA3A），人密码子优化的nCas9(D10A)，和K.phafii密码子优化的尿嘧啶糖基化酶抑制剂(KpUGI)。这个最佳的基础编辑器在K.phafii中有效地执行了C到T编辑，与单-，double-,三基因座编辑效率高达96.0%，65.0%,和5.0%，分别,在从C-18到C-12的7个核苷酸窗口内。为了扩大可靶向基因组区域，我们还用nSpG和nSpRy替换了最佳基础编辑器中的nCas9，NGN-前间隔区相邻基序(PAM)位点的C-T编辑效率达到50.0%-60.0%,NRN-PAM位点的C-T编辑效率达到20.0%-93.2%,分别。因此,这些构建的基础编辑器已经成为基因功能研究的强大工具，代谢工程,基因改良,以及K.phafii的功能基因组学研究。
