e14688 Background: Functional analysis of the multitude of mutations found in tumors is a major goal to better understand their role and to optimize patient treatment. PCR-based site-directed mutagenesis (SDM) techniques are often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability and lack of multiplexing capabilities. To overcome many of these limitations, we describe a novel, fast and simple method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro CRISPR based DNA editing. Methods: For each mutation, a specifically designed pair of CRISPR/Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA enabling the excision of a defined DNA fragment. This is followed by donor DNA replacement and bacterial colony expansion. We term this method, CRISPR-directed DNA Mutagenesis (CDM). Results: Using CDM we have been able to synthesize known oncogenic mutations as well as novel variants in 8 different cancer genes. These mutations have been synthesized with over 60% success rate, compared to about 40% success rate in SDM. More importantly, we show that in the CDM method there were no off-site mutations eliminating the need to sequence large portions of the gene. Conclusions: We have developed a novel multiplex site-directed mutagenesis method that can generate multiple unique mutations simultaneously within plasmids. CDM has proven capable of precise, rapid and robust mutation synthesis, including single base point mutations, site-specific deletions, insertions and duplications within targeted plasmids.
A simple and efficient method for site-directed mutagenesis with double-stranded plasmid DNA
