PDF(Pubmed)
Abstract：
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 Komagataella phaffii, 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 K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), 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 K. phaffii. 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%).
摘要：
基于CRISPR（成簇的规则间隔的短回文重复）的技术非常强大，用于定点基因组修饰的可编程工具。在成功适应并有效使用CRISPR-Cas9对甲基营养酵母Komagataellaphafii进行基因组工程后，更广泛的可使用的核酸内切酶被期望以增加实验灵活性并且在由于第三方的知识产权(IPR)而在工业研究中存在特定法律限制的情况下提供替代方案。MAD7，一种工程化的2类VCas核酸酶，被推广为学术和工业研究的免版税替代品，并由Inscripta(Pleasanton，CA,美国）。在这项研究中,第一次,CRISPR-MAD7用于K.phafii的基因组编辑，具有很高的基因编辑率（高达90％），正如编码甘油激酶1（GUT1）的三个靶向基因所证明的那样，红色荧光蛋白（DsRed），和zeocin抗性基因(Shble)。此外,通过靶向K.phafii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.在这个广泛的测试中，CRISPR-Cas9的基因组编辑率更高，约为65%，与应用的CRISPR-MAD7工具箱相比（约23%）。
