Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.
Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.
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DOI：
10.1128/spectrum.02321-21
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发表时间：
2022-06-29
影响因子：
3.7
通讯作者：
Tian, Chaoguang
中科院分区：
文献类型：
--
作者：
Zhang, Chenyang;Li, Nan;Rao, Lang;Li, Jingen;Liu, Qian;Tian, Chaoguang
关键词：
嗜热毁丝霉（Myceliophthora thermophila）是一种在生物炼制和生物技术领域具有巨大潜力的嗜热真菌。碱基编辑器是成簇规则间隔短回文重复序列（CRISPR）依赖性基因组编辑工具的升级版本，该工具引入精确的点突变而不会导致DNA双链断裂（DSB），已用于各种生物体，但很少用于丝状真菌，特别是嗜热丝状真菌。在此，我们首次在M.嗜热菌，即进化的载脂蛋白B mRNA编辑酶催化亚基1（APOBEC 1）胞嘧啶碱基编辑器4 max（Mtevo-BE 4 max）、噬菌体Mu Gam蛋白胞嘧啶碱基编辑器4 max（MtGAM-BE 4 max）和进化的CDA 1脱氨酶胞嘧啶碱基编辑器（Mtevo-CDA 1），并通过精确地将三个密码子（CAA、CAG和CGA）转化为终止密码子而不形成DSB来有效地灭活基因。具有高达92.6%编辑效率的Mtevo-CDA 1编辑器是嗜热真菌中胞嘧啶碱基编辑的更合适的工具。研究纤维素酶转录因子M的各个基序的功能。为了鉴定嗜热菌Mtagen-2（Mtagen-2），我们使用Mtevo-CDA 1编辑器。真菌特异性基序的Mtagel-2被发现强烈参与纤维素酶分泌，分生孢子形成，菌丝分支，和菌落形成。真菌特异性基序的突变在这些特征中造成了显着的缺陷。因此，我们开发了一种高效的嗜热真菌相容性碱基编辑系统，该系统也可用于其他相关丝状真菌的基因工程。一种基于CRISPR/Cas的碱基编辑方法已经开发出来，可以在不诱导双链断裂（DSB）的情况下引入点突变，并引起了学术界和工业界的极大兴趣。我们的研究开发了脱氨酶-胞嘧啶碱基编辑系统，有效地编辑三个目标基因，amdS，cre-1，和必需的纤维素酶调节基因Mtclr-2，在嗜热毁丝霉。在M的DNA结合域和真菌特异性基序的靶位点上发现了多种点突变。通过我们的碱基编辑器Mtevo-CDA 1成功地产生了嗜热菌Mtevo-2（Mtevo-2），以阐明其功能。在这里，我们表明，Mtagene-2的DNA结合结构域是重要的真菌响应纤维素条件下，而其真菌特异性基序参与真菌生长。这些发现表明，我们的碱基编辑器可以是一个有效的工具，阐明丝状真菌中的目标基因的基序的功能和合成生物学领域的代谢工程。
Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi. IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.
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