CRISPR/Cas systems have undergone tremendous advancement up to now decade. These precise genome editing tools have applications starting from transgenic crop development to gene therapy and beyond. And with their recent development of CRISPR-COPIES, researchers on the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) are further improving CRISPR’s versatility and ease of use.
“CRISPR-COPIES is a tool that may quickly discover appropriate chromosomal integration sites for genetic engineering in any organism,” said Huimin Zhao, CABBI Conversion Theme Leader and Steven L. Miller Chair of Chemical and Biomolecular Engineering (ChBE) on the University of Illinois. “It is going to speed up our work within the metabolic engineering of non-model yeasts for cost-effective production of chemicals and biofuels.”
Gene editing has revolutionized scientists’ capabilities in understanding and manipulating genetic information. This type of genetic engineering allows researchers to introduce latest traits into an organism, comparable to resistance to pests or the power to supply a beneficial biochemical.
With CRISPR/Cas systems, researchers could make precise, targeted genetic edits. Nevertheless, locating optimal integration sites within the genome for these edits has been a critical and largely unsolved problem. Historically, when researchers needed to find out where to focus on their edits, they might typically manually screen for potential integration sites, then test the location by integrating a reporter gene to evaluate its cellular fitness and gene expression levels. It is a time- and resource-intensive process.
To handle this challenge, the CABBI team developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool can discover genome-wide neutral integration sites for many bacterial and fungal genomes inside two to 3 minutes.
“Finding the combination site within the genome manually is like looking for a needle in a haystack,” said Aashutosh Boob, a ChBE Ph.D. student on the University of Illinois and first creator of the study. “Nevertheless, with CRISPR-COPIES, we transform the haystack right into a searchable space, empowering researchers to efficiently locate all of the needles that align with their specific criteria.”
Of their paper published in Nucleic Acids Research, the researchers demonstrated the flexibility and scalability of CRISPR-COPIES by characterizing integration sites in three diverse species: Cupriavidus necator, Saccharomyces cerevisiae, and HEK 293T cells. They used integration sites found by CRISPR-COPIES to engineer cells with increased production of 5-aminolevulinic acid, a beneficial biochemical that has applications in agriculture and the food industry.
As well as, the team has created a user-friendly web interface for CRISPR-COPIES. This incredibly accessible application might be utilized by researchers even without significant bioinformatics expertise.
A primary objective of CABBI is the engineering of non-model yeasts to supply chemicals and fuels from plant biomass. Economically producing biofuels and bioproducts from low-cost feedstocks at a big scale is a challenge, nevertheless, because of the dearth of genetic tools and the cumbersome nature of traditional genome-editing methods. By enabling researchers to swiftly pinpoint genomic loci for targeted gene integration, CRISPR-COPIES provides a streamlined pipeline that facilitates the identification of stable integration sites across the genome. It also eliminates the manual labor involved in designing components for CRISPR/Cas-mediated DNA integration.
For crop engineering, the tool might be used to extend biomass yields, pest resistance, and/or environmental resilience. For converting biomass to beneficial chemicals — for example, by utilizing the yeast S. cerevisiae — CRISPR-COPIES might be used to engineer cells with significantly greater yields.
This versatile software is designed to simplify and speed up the strain construction process, saving researchers each time and resources. Researchers all over the world in each academia and industry can profit from its utility in strain engineering for biochemical production and transgenic crop development.
Co-authors on this study include ChBE Ph.D. student Zhixin Zhu, ChBE visiting student Pattarawan Intasian, and Bioengineering Ph.D. student Guanhua Xun; Carl R. Woese Institute for Genomic Biology (IGB) Software Developers Manan Jain and Vassily Petrov; IGB Biofoundry Manager Stephan Lane; and CABBI postdoc Shih-I Tan.
— Article by CABBI Communications Specialist April Wendling
