CRISPR-copies: New tool accelerates and optimizes genome editing
CABBI researchers publically share a new tool to revolutionize CRISPR gene editing
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN INSTITUTE FOR SUSTAINABILITY, ENERGY, AND ENVIRONMENT
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CRISPR-COPIES HAS APPLICATIONS IN SYNTHETIC BIOLOGY TOOLKIT CHARACTERIZATION, GENE THERAPY, AND METABOLIC ENGINEERING.
view moreCREDIT: AASHUTOSH BOOB ET AL.
CRISPR/Cas systems have undergone tremendous advancement in the past decade. These precise genome editing tools have applications ranging from transgenic crop development to gene therapy and beyond. And with their recent development of CRISPR-COPIES, researchers at 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 can quickly identify 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) at the University of Illinois. “It will accelerate our work in 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 form of genetic engineering allows researchers to introduce new traits into an organism, such as resistance to pests or the ability to produce a valuable biochemical.
With CRISPR/Cas systems, researchers can make precise, targeted genetic edits. However, locating optimal integration sites in the genome for these edits has been a critical and largely unsolved problem. Historically, when researchers needed to determine where to target their edits, they would typically manually screen for potential integration sites, then test the site by integrating a reporter gene to assess its cellular fitness and gene expression levels. It’s a time- and resource-intensive process.
To address this challenge, the CABBI team developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool can identify genome-wide neutral integration sites for most bacterial and fungal genomes within two to three minutes.
“Finding the integration site in the genome manually is like searching for a needle in a haystack,” said Aashutosh Boob, a ChBE Ph.D. student at the University of Illinois and primary author of the study. “However, with CRISPR-COPIES, we transform the haystack into a searchable space, empowering researchers to efficiently locate all the needles that align with their specific criteria.”
In their paper published in Nucleic Acids Research, the researchers demonstrated the versatility 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 valuable biochemical that has applications in agriculture and the food industry.
In addition, the team has created a user-friendly web interface for CRISPR-COPIES. This incredibly accessible application can be used by researchers even without significant bioinformatics expertise.
A primary objective of CABBI is the engineering of non-model yeasts to produce chemicals and fuels from plant biomass. Economically producing biofuels and bioproducts from low-cost feedstocks at a large scale is a challenge, however, due to the lack 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 can be used to increase biomass yields, pest resistance, and/or environmental resilience. For converting biomass to valuable chemicals — for instance, by using the yeast S. cerevisiae — CRISPR-COPIES can be used to engineer cells with significantly greater yields.
This versatile software is designed to simplify and accelerate the strain construction process, saving researchers both time and resources. Researchers around the world in both academia and industry can benefit 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
JOURNAL
Nucleic Acids Research
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Not applicable
ARTICLE TITLE
CRISPR-COPIES: An in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration
ARTICLE PUBLICATION DATE
13-Feb-2024
Not too late to repair: gene therapy improves advanced heart failure in animal model
Heart failure remains the leading cause of mortality in the U.S. During a heart attack blood stops flowing into the heart. Without oxygen, part of the heart muscle dies. The heart muscle does not regenerate, instead it replaces dead tissue with a scar made of cells called fibroblasts that do not help the heart pump. If there is too much scarring, the heart progressively enlarges, or dilates, weakens and eventually stops working.
“The current thought is that advanced or chronic heart failure, a stage in which the cardiac muscle has become too weak, is a point of no return. The present understanding is that it is not possible to stimulate a heart in this condition to generate new heart cells to repair itself and that only palliative treatment is available to patients,” said corresponding author Dr. Tamer M. A. Mohamed, associate professor of surgery and medicine and director of cardiac regeneration at Baylor College of Medicine. “In this study published in the journal Cardiovascular Research, we show that advanced heart failure can be treated to improve cardiac function in an animal model.”
In a previous study, Mohamed and his collaborators had successfully used gene therapy to improve acute cardiac dysfunction in animals. Their method effectively and specifically delivered genes that promote proliferation to heart cells, generating new heart muscle. This approach not only strengthened the heart improving its ability to keep the blood flowing, but also prevented typical subsequent congestion in the liver, kidneys and lungs in rats and pigs.
“In this study, we did something that had not been done before,” Mohamed said. “We intervened with the same gene therapy but not during acute heart failure or early in the disease as in our previous experiments, but late in the disease during the chronic phase four weeks after cardiac injury had severely damaged the heart.”
Four months after treating the animals, the researchers checked cardiac function and heart structure. “We were surprised to see evidence of significant heart cell proliferation, a marked reduction in scar size and a significant improvement in cardiac function,” said first author Dr. Riham R E Abouleisa, assistant professor of surgery- cardiothoracic surgery at Baylor. “Although heart dilation and lung congestion associated with chronic heart failure were not improved, the treatment partially improved liver and kidney functionality.”
The findings show for the first time that contrary to expectations, it is possible to induce heart cell proliferation during advanced states of heart failure and improve heart function, with some beneficial effects on the liver and kidneys’ functions.
“Our work has important implications for the large group of patients with advanced heart failure for whom there are currently no treatments to improve their condition,” Mohamed said. “This approach offers the possibility of developing future new therapies for this deadly disease.”
Other authors on this study include: Xian-Liang Tang, Qinghui O, Abou-Bakr M. Salama, Amie Woolard, Dana Hammouri, Hania Abdelhafez, Sarah Cayton, Sameeha K. Abdulwali, Momo Arai, Israel D. Sithu, Daniel J. Conklin and Roberto Bolli. The authors are affiliated with one or more of the following institutions: Baylor College of Medicine, University of Louisville, Zagazig University, Alfaisal University and University of Manchester.
This work was supported by the National Institutes of Health grants (F32HL149140, R01HL147921, P30GM127607, R15HL168688, R01HL166280 and HL78825) and by the American Heart Association grant 16SDG29950012.
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JOURNAL
Cardiovascular Research
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Animals
ARTICLE TITLE
Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats
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