DRIMS: A Synthetic Biology Platform that Enables Deletion, Replacement, Insertion, Mutagenesis, and Synthesis of DNA

Abstract

DNA modification and synthesis are fundamental to genetic engineering, and systems that enable time- and cost-effective execution of these processes are crucial. Iteration of genetic construct variants takes significant time, cost and effort to develop new therapeutic strategies to treat diseases including cancer. Thus, decreasing cost and enhancing simplicity while accelerating the speed of advancement is critical. We have developed a PCR-based platform that allows for deletion, replacement, insertion, mutagenesis, and synthesis of DNA (DRIMS). These modifications rely on the recA-independent recombination pathway and are carried out in a single amplification step followed by DpnI digestion and transformation into competent cells. DNA synthesis is accomplished through sequential PCR amplification reactions without the need for a DNA template. Here, we provide proof-of-concept for the DRIMS platform by performing four deletions within DNA fragments of various sizes, sixty-four replacements of DNA binding sequences that incorporate repeat sequences, four replacements of chimeric antigen receptor components, fifty-one insertions of artificial microRNAs that form complex tertiary structures, five varieties of point mutations, and synthesis of eight DNA sequences including two with high GC content. Compared to other advanced cloning methods including Gibson and “in vivo assembly”, we demonstrate the significant advantages of the DRIMS platform. In summary, DRIMS allows for efficient modification and synthesis of DNA in a simple, rapid and cost-effective manner to accelerate the synthetic biology field and development of therapeutics.