Based on the CRISPR-Cas9 technology, scientists from Denmark develop Cas9-mediated protein evolution reaction (CasPER) to build a library of diverse enzymes, which may be used in the industry and academe. The methodology involves the usage of error-prone PCR (epPCR) and CRISPR-Cas9-mediated homology-directed repair (HDR) to edit the DNA of targeted enzymes and produce variants.

Scientist Jay Keasling and colleagues from The Novo Nordisk Foundation Center for Biosustainability in Denmark utilized epPCR to generate different DNA sequences for insertion via HDR to the CRISPR-Cas9-generated cut site in the enzyme DNA. The team tested the method in Saccharomyces cerevisiae by targeting two essential enzymes in the mevalonate pathway. Results showed 11-fold increase in the expression of targeted enzymes, and sequencing confirmed the integrity of the inserted DNA. CasPER also shows to have an editing efficiency of more than 98 percent.

CasPER allows for simultaneous engineering of several enzyme variants and large-fragment inserts. It may be used in other experiments in the industry and the academe.

For more information, read the research article in Metabolic Engineering.