Spotting Every Needle in the Haystack: CRISPeR Gene Editing for DNA with Lone Mutations

SEOUL, Jan. 25, 2021 /PRNewswire/ -- The quest to change the very nature of being, has driven philosophical and metaphysical debates for ages. Now, with the advent of genome editing techniques, man is changing the very fabric of biological existence quite literally. One revolutionary tool in the genome editing toolbox is the CRISPR-Cas9 system.

There are two key molecules in this system: A single-molecular guide RNA (sgRNA) that binds to foreign DNA, and an enzyme, called Cas9, which acts as molecular scissors to cleave the targeted DNA sequence flagged by specific genomic landmarks. Scientists have used this gene editing tool in combination with techniques to introduce 2–3 specific mutations at chosen locations in the genome of microbial cells to obtain desired phenotypes for scientific and industrial application. However, till date they have not been able to successfully obtain cells with mutations in single 'bases' or building blocks of the target DNA.

In a recent study published in Genome Research, a team at Chung-Ang University, Korea, has now found a way to solve this issue. Describing their unique technique, Dr. Sang Jun Lee, who led the study, says, "Our team upgraded and developed a single base editing method using target-mismatched sgRNAs of the CRISPR-Cas9 system."

What he means is, they dealt with the problem of recognizing lone mismatched bases in a DNA strand by introducing a mismatch in the guide RNA of the CRISPR-Cas9 system. Starting with 48 desired single-base mutations in their sgRNA, the scientists successfully edited 25 single bases across 16 target sequences in the E. coli genome. Overall, they introduced single-point base mutations in the galK gene of 36%−95% of E. coli cells, a massive improvement over the previously achieved efficiency of 3%.

The total number of single base mutations in a microbe's genome can number in the thousands and a gene editing tool that allows for the selection of cells with such unique and diverse mutations could be the ultimate gamechanger in the genetic engineering industry. As Prof. Lee observes, "Our single base editing method can be used to engineer precise recombinant microbial strains that are tailored to meet individual patient's requirements. By improving existing microbiome manipulation technologies, our technique will ultimately improve the quality of public health."

Microbial cells are our source of pharmacologically important biomolecules, and scientists strive daily to alter the microbial genome to develop drugs of choice, for diseases with a genetic basis and particularly in this age of personalized medicine. This technique could be the latest addition to the vast repertoire of tools by which humanity hopes to literally shape its destiny.

Reference

Title of original paper: CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

Journal: Genome Research

DOI: http://www.genome.org/cgi/doi/10.1101/gr.257493.119

Original Article: https://bit.ly/3c29yqS

Chung-Ang University Website

https://neweng.cau.ac.kr/index.do 

About Professor Sang Jun Lee

Prof. Sang Jun Lee is an Associate Professor of Department of Systems Biotechnology at Chung-Ang University, Korea. He works on CRISPR/Cas based bacterial genome editing tools and systems metabolic biotechnology. Before joining Chung-Ang University, he was the Chief of the Microbiomics and Immunity Research Center at the Korea Research Institute of Bioscience and Biotechnology. Prof. Lee received his PhD from the Department of Biological Sciences at KAIST (Korea Advanced Institute of Science and Technology) and completed his postdoctoral training from the National Institutes of Health, Bethesda, Maryland. He has also been the Deputy Editor of the Journal of Microbiology and Biotechnology from 2018 to 2020.

Contact:
Seong-Kee Shin
02-820-6614
[email protected]

SOURCE Chung-Ang University