Scientists at the CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB) in New Delhi have created a new genome-editing system that is better and more precise than the current CRISPR technologies.
Understanding CRISPR
CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a natural defense system in bacteria that destroys viral DNA. Scientists have adapted this system to change the genes of more complex organisms.
How CRISPR-Cas9 Works
The CRISPR-Cas9 technology allows scientists to add, remove, or change DNA sequences. It uses a guide RNA (gRNA) to direct the Cas9 enzyme to a specific DNA location, where it makes a cut. The cell then repairs this cut, which can fix or change the DNA. However, the traditional CRISPR-Cas9 system can sometimes unintentionally affect other parts of the genome, known as “off-target” effects. Even though scientists have made improvements to make it more accurate, these changes often reduce how well it works.
Introducing FnCas9
To address this problem, researchers have been studying the FnCas9 enzyme from the bacteria Francisella novicida. This enzyme is more precise and efficient than the SpCas9 variant but usually less effective. The CSIR-IGIB team improved FnCas9 by altering its amino acid interactions with the PAM sequence. This change increased its ability to bind to DNA and made gene editing more effective. These modifications also helped the enzyme reach difficult-to-access areas of the genome.
Better Diagnostic Capabilities
Experiments showed that the improved FnCas9 could identify specific single-nucleotide changes in DNA more efficiently than the unmodified version, doubling its ability to detect genetic variations related to diseases. The enhanced FnCas9 was tested on human kidney and eye cells and showed higher precision with minimal off-target effects compared to SpCas9. This highlights its potential for treating genetic disorders.
Case Study: Leber Congenital Amaurosis 2 (LCA2)
The improved FnCas9 enzyme was used to fix a mutation in the RPE65 gene, which causes a type of blindness called Leber Congenital Amaurosis 2 (LCA2). The editing almost completely corrected the mutation and led to normal protein production in retinal cells. This method worked better than older systems. Because of these promising results, scientists now want to explore using stem cells from individual patients. They plan to edit these stem cells to fix mutations before transplanting them back into the patient. This approach could be safer and more precise than directly using CRISPR on patients.
