Mousavi Negar, Zhou Ethan, Razavi Arezousadat, Ebrahimi Elham, Varela-Castillo Paulina, Yang Xiang-Jiao
Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, Quebec, Canada; Department of Medicine, McGill University, Montreal, Quebec, Canada.
Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, Quebec, Canada; Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 2025 Mar;301(3):108219. doi: 10.1016/j.jbc.2025.108219. Epub 2025 Jan 23.
Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the ideal efficiency of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3'-overhangs, but this approach has not been fully developed. As the first step toward reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases, and modification of PCR parameters) and evaluated the resulting method extensively with >100 mutations on 12 mammalian expression vectors, ranging from 7.0 to 13.4 kb in size and encoding ten epigenetic regulators linked to cancer and neurodevelopmental disorders. We have also tested the new method with two expression vectors for the SARS-CoV-2 spike protein. Compared to the QuickChange method, the success rate has increased substantially, with an average efficiency of ∼50%, with some at or close to 100%, and requiring much less time for engineering various mutations. Therefore, we have developed a new site-directed mutagenesis method for efficient and economical generation of various mutations. Notably, the method failed with a human KAT2B expression plasmid that possesses extremely GC-rich sequences. Thus, this study also sheds light on how to improve the method for developing ideal mutagenesis methods with the efficiency of ∼100% for a wide spectrum of plasmids.
定点诱变是蛋白质和质粒工程中不可或缺的基本工具。一个重要的技术问题是如何实现100%的理想效率。基于互补引物对的QuickChange方法已被广泛使用，但由于其效率低且频繁出现不需要的突变，需要大幅改进。一种替代的创新策略是使用带有3'突出端的引物对，但这种方法尚未得到充分发展。作为实现100%效率的第一步，我们系统地优化了这种方法（如新设计的短引物的使用、不同Pfu DNA聚合酶的测试以及PCR参数的修改），并在12个哺乳动物表达载体上对产生的方法进行了广泛评估，这些载体大小从7.0到13.4 kb不等，编码与癌症和神经发育障碍相关的十种表观遗传调节因子。我们还用两种用于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的表达载体测试了这种新方法。与QuickChange方法相比，成功率大幅提高，平均效率约为50%，有些达到或接近100%，并且对各种突变进行工程改造所需的时间要少得多。因此，我们开发了一种新的定点诱变方法，用于高效、经济地产生各种突变。值得注意的是，该方法在具有极高GC含量序列的人KAT2B表达质粒上失败了。因此，本研究也为如何改进该方法以开发适用于广泛质粒的、效率约为100%的理想诱变方法提供了思路。
