New toolbox allows engineering of genomes without CRISPR

Belgian researchers from the VIB-KULeuven Center for Microbiology and the VIB-UGent Center for Plant Systems Biology have developed a new toolbox of 16 different short DNA sequences that allow the triggering of controlled and specific maintenance events in any genome. Gives.

This new patent toolbox, complementing—and for some applications beyond—CRISPR, is now available to researchers and industry in the field of genome engineering. The results are reported today in two concurrent papers. Nature Communications.

Site-specific recombinases enable efficient cutting and pasting of DNA at specific locations in the genome, where each recombinase recognizes a precise DNA sequence. Because of their sequence specificity, CRISPR systems have superseded site-specific recombination as a genome engineering tool in the past decade.

CRISPR systems have revolutionized the field because they can be easily targeted to different genomic loci. However, because site-specific recombinases work differently, they avoid some of the major problems of CRISPR, including the toxicity of DNA double-strand breaks leading to unwanted point mutations and structural mutations, many of which are unconventional. Organisms face low efficacy of modification. Difficulty with cell division and insertion of large DNA fragments. Additionally, CRISPR’s very complex patents make its use in research and industry often difficult and expensive.

Research groups at the VIB-KULeuven Center for Microbiology and their colleagues at the VIB-UGent Center for Plant Systems Biology have now overcome the shortcomings of earlier site-specific recombination as a tool for genomic engineering. The team has expanded the toolbox that uses viral recombinase (Cre) so that it can now specifically recognize, cut and paste multiple DNA sites. The team identified a set of 16 sites that efficiently recombined with a single site, but not with any other site in that set, and for different organisms.

“With these orthogonal recombination systems, we can avoid the unpredictable way in which multiple recombination sites in a genome interact with each other,” says Kevin Verstrepen, director of the VIB-KULeuven Center for Microbiology. ” “This opens the way for many research projects to install several small or large genomic edits simultaneously or to repeatedly recycle markers during genome engineering efforts.”

Applications to research and industry.

Charlotte Cauteriels, Ph.D. student in Kevin Verstripen’s lab set up and tested the new toolbox in yeast. Bacterial cells. He then collaborated with the VIB-UGent PSB Center to demonstrate its performance in plant cells as well.

In her latest publication, she demonstrates how it can be used to optimize the expression of metabolic pathway genes and production titers of industrially relevant molecules. A single round of shuffling dedicated gene regulators with new recombination sites already doubled production titers.

“We were able to confirm that modifying expression through our recombination-based setup allows rapid and efficient optimization of gene expression in heterologous biosynthetic pathways,” says Cautereels. “This new toolbox not only offers the potential to improve microbial cell factories, but also validates our toolbox for scientists and industry alike. The genome Engineering.”