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.