Smirnov Kirill, Weiss Florian, Hatzl Anna-Maria, Rieder Lukas, Olesen Kjeld, Jensen Sanne, Glieder Anton
Christian Doppler Laboratory for Innovative Pichia pastoris Host and Vector Systems, Institute of Molecular Biotechnology, Graz University of Technology, 8010 Graz, Austria.
Novo Nordisk A/S, Novo Nordisk Park, DK 2760 Måløv, Denmark.
J Fungi (Basel). 2024 Mar 5;10(3):197. doi: 10.3390/jof10030197.
CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast , a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (), red fluorescence protein (), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in . In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).
基于CRISPR（成簇规律间隔短回文重复序列）的技术是用于定点基因组修饰的强大的、可编程工具。在成功将CRISPR-Cas9应用于甲基营养型酵母的基因组工程并高效使用之后，人们期望有更多种类的可用核酸内切酶，以增加实验灵活性，并在工业研究中因第三方知识产权（IPR）存在特定法律限制的情况下提供替代方案。MAD7是一种经过改造的2类V型Cas核酸酶，被推广为学术和工业研究的免版税替代方案，由美国加利福尼亚州普莱森顿的Inscripta公司开发。在本研究中，首次将CRISPR-MAD7用于[具体物种名称缺失]的基因组编辑，基因编辑率高（高达90%），编码甘油激酶1（[具体基因名称缺失]）、红色荧光蛋白（[具体基因名称缺失]）和博来霉素抗性基因（Sh ble）的三个靶向基因的情况就是证明。此外，通过靶向[具体物种名称缺失]中的259个激酶基因，系统地比较了CRISPR-MAD7和CRISPR-Cas9系统的基因组编辑效率。在这项广泛的测试中，与应用的CRISPR-MAD7工具箱（约23%）相比，CRISPR-Cas9的基因组编辑率更高，约为65%。
