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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.

DOI: 10.1021/acssynbio.4c00649
PMID: 39902634