Rapid and Error-Free Site-Directed Mutagenesis by a PCR-Free In Vitro CRISPR/Cas9-Mediated Mutagenic System,ACS Synthetic Biology

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Rapid and Error-Free Site-Directed Mutagenesis by a PCR-Free In Vitro CRISPR/Cas9-Mediated Mutagenic System
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-08-03 00:00:00 , DOI: 10.1021/acssynbio.8b00245
Wenwen She ₁ , Jing Ni ₁ , Ke Shui ₂ , Fei Wang ₁ , Ruyi He ₁ , Jinhui Xue ₁ , Manfred T. Reetz _{3,

4} , Aitao Li ₁ , Lixin Ma ₁

Affiliation

The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, among other things, amino acid bias, which means that the development of efficient PCR-free methods is desirable. Here, we present a highly efficient in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants in a PCR-free manner. First, it involves plasmid digestion by utilizing a complex of Cas9 with specific single guide RNA (sgRNA) followed by degradation with T5 exonuclease to generate a 15 nt homologous region. Second, primers containing the desired mutations are annealed to form the double-stranded DNA fragments, which are then ligated into the linearized plasmid. In theory, neither the size of the target plasmid nor the unavailable restriction enzyme site poses any problems that may arise in traditional techniques. In this study, single and multiple site-directed mutagenesis were successfully performed even for a large size plasmid (up to 9.0 kb). Moreover, a PCR-free site-saturation mutagenesis library on single site and two adjacent sites of a green fluorescent protein was also generated with promising results. This demonstrates the great potential of the ICM system for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.

中文翻译：

通过无PCR的体外CRISPR / Cas9介导的诱变系统进行快速，无错误的定点诱变

除其他因素外，由于氨基酸偏倚，任何基于PCR的诱变技术的质量和效率都可能不是最佳的，这意味着需要开发高效的无PCR方法。在这里，我们提出了一种高效的体外CRISPR / Cas9介导的诱变（ICM）系统，允许以无PCR的方式快速构建设计的突变体。首先，它涉及通过利用Cas9与特定单向导RNA（sgRNA）的复合物进行质粒消化，然后用T5核酸外切酶降解以产生15 nt同源区域。其次，将含有所需突变的引物退火以形成双链DNA片段，然后将其连接到线性化质粒中。从理论上讲，靶质粒的大小和不可用的限制酶位点都不会造成传统技术中可能出现的任何问题。在这项研究中，即使对于大质粒（最大9.0 kb），也成功地进行了单点和多点诱变。而且，还产生了绿色荧光蛋白的单个位点和两个相邻位点的无PCR的位点饱和诱变文库，具有可喜的结果。这证明了ICM系统在定向进化中创建高质量的突变文库作为基于PCR的饱和诱变的替代方法的巨大潜力，从而促进了合成生物学的研究。

更新日期：2018-08-03

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