DEC 04, 2020 5:46 PM PST

An 'Off' Switch to Improve CRISPR Precision

WRITTEN BY: Carmen Leitch

The CRISPR-Cas9 gene-editing tool has tremendous potential to treat diseases that are caused by genetic errors. The idea behind it is to bring a pair of ‘molecular scissors’ to a very specific place in the genome (such as where a disease-causing mutation sits) to make a cut and remove an erroneous sequence. A DNA fragment containing the correct sequence can then be joined to the genome, and in theory, the mutation causing the disease would be repaired, and the symptoms of the disease would be cured. But sometimes CRISPR-Cas9 makes erroneous cuts at unintended places. These so-called off-target effects can have major consequences, impeding the potential of the gene editor.

Image credit: Modified from Pixabay/french1944

Now researchers have developed a tool to stop these unintended changes, which works to control CRISPR-Cas9 so it’s only active during a certain period in a cell’s cycle. The activity of the editor is meant to be limited to periods when gene editing is more likely to be accurate. While other, similar methods have been devised, this one aims to make CRISPR-Cas9 more precise without reducing its efficacy. The work has been reported in Communications Biology.

"We aimed to develop the method to avoid the side effect called off-target effect which is one of the most challenging problems in the genome-editing field," said study author Wataru Nomura, a professor at Hiroshima University's Graduate School of Biomedical and Health Sciences. "Our method is like hitting two birds with one stone. We can improve the preciseness of genome editing and suppression of off-target effects at the same time."

In this work, the researchers utilized an 'anti-CRISPR' protein called AcrIIA4 that counteracts the action of a version of Cas9 called SpyCas9, and could act as an off-switch for CRISPR. The scientists combined AcrIIA4 with the terminus of another protein that ensures the genome is only copied once during the cell cycle: Cdt1. The AcrIIA4-Cdt1 fusion protein seems to restrict CRISPR-Cas9 gene-editing to the S and G2 cell cycle phases, which is when a process called homology-directed repair (HDR) is more likely to occur.

When there are breaks in DNA, the cell has to repair them, and it has two ways to do so, called non-homologous end-joining (NHEJ) and HDR. Most chromosomes come in pairs, and HDR uses the second chromosome as a template to repair the break in the first. However, NHEJ usually just brings broken ends of DNA back together at any stage of the cell cycle, while HDR is more precise and is restricted to fewer stages.

The levels of ArIIA4-Cdt1 fusion were found to depend on the cell cycle, increasing during G1 phase and stopping NHEJ from making repairs. AcrIIA4-Cdt1 levels go down during subsequent phases.

With the fusion protein, the researchers reduced off-target mutations by about 86.5 percent in one test, and from 8.5 percent to 0.6 percent in another test. By co-expressing SpyCas9 and AcrIIA4-Cdt1, the researchers were able to increase HDR frequency while suppressing off-target effects.

"We envision to apply our system to other CRISPR/anti-CRISPR combinations as well as other CRISPR based gene editor such as base editors and targeted transcription mediators," said Nomura. "Our ultimate goal is to develop a genome editing system which can be used safely in the medical therapeutic field."

Sources: AAAS/Eurekalert! via Hiroshima University, Communications Biology

About the Author
  • Experienced research scientist and technical expert with authorships on over 30 peer-reviewed publications, traveler to over 70 countries, published photographer and internationally-exhibited painter, volunteer trained in disaster-response, CPR and DV counseling.
You May Also Like
FEB 24, 2021
Microbiology
The Human Gut Microbiome is Teeming With Viruses
FEB 24, 2021
The Human Gut Microbiome is Teeming With Viruses
Using metagenomics tools, which sequence all of the viral material in a sample, researchers have investigated the viruse ...
MAR 02, 2021
Genetics & Genomics
Human Genome Sequencing Reveals Surprising Level of Structural Variation
MAR 02, 2021
Human Genome Sequencing Reveals Surprising Level of Structural Variation
It was a landmark event when the human genome was finally sequenced, and the Human Genome Project came to an end. But li ...
MAR 11, 2021
Genetics & Genomics
50 New Eye Color Genes Are Identified
MAR 11, 2021
50 New Eye Color Genes Are Identified
The genetics of eye color are far more complex than researchers once thought.
MAR 22, 2021
Microbiology
A Cavity-Forming Germ Gets a Little Help From a Gene Cluster
MAR 22, 2021
A Cavity-Forming Germ Gets a Little Help From a Gene Cluster
There are many microbes that live in the human mouth, and while some are harmless, others like Streptococcus mutans are ...
MAR 28, 2021
Cell & Molecular Biology
Understanding How Cold-Induced Tooth Pain Happens
MAR 28, 2021
Understanding How Cold-Induced Tooth Pain Happens
Our teeth do a lot of work, and they may become sensitive to cold as the gums erode due to aging or because they have an ...
APR 14, 2021
Genetics & Genomics
The Smell of Home: Insights Into Scent Imprinting
APR 14, 2021
The Smell of Home: Insights Into Scent Imprinting
Right after birth, young animals go through a period in which they 'imprint,' or fixate on sights and smells that they'r ...
Loading Comments...