Safer CRISPR gene editing with fewer off-target hits

Date:
July 9, 2020
Source:
PLOS
Summary:
The CRISPR system is a powerful tool for the targeted editing of
genomes, with significant therapeutic potential, but runs the
risk of inappropriately editing ''off-target'' sites. However,
a new study shows that mutating the enzyme at the heart of the
CRISPR gene editing system can improve its fidelity.



FULL STORY ==========================================================================
The CRISPR system is a powerful tool for the targeted editing of
genomes, with significant therapeutic potential, but runs the risk
of inappropriately editing "off-target" sites. However, a new study
publishing July 9, 2020 in the open- access journal PLOS Biology by
Feng Gu of Wenzhou Medical University, China, and colleagues, shows that mutating the enzyme at the heart of the CRISPR gene editing system can
improve its fidelity. The results may provide a therapeutically safer
strategy for gene editing than using the unmodified enzyme system.


==========================================================================
The CRISPR system employs an enzyme called Cas9 to cleave DNA. Cas9 will
cut almost any DNA sequence. Its specificity comes from its interaction
with a "guide RNA" (gRNA) whose sequence allows it to bind with the
target DNA through base-pair matching. Once it does, the enzyme is
activated and the DNA is cut.

The CRISPR system is found in multiple bacterial species; among those
commonly used in research, that from Staphylococcus aureus has the
advantage of size - - unlike some others, its gene is small enough
to fit inside a versatile and harmless gene therapy vector called adeno-associated virus, making it attractive for therapeutic purposes.

A key limitation of any of the CRISPR systems, including that from
S. aureus, is off-target cleavage of DNA. A guide RNA may bind weakly
to a site whose sequence is a close but imperfect match; depending on
how close the match is and how tightly the enzyme interacts with the
paired gRNA-DNA complex, the enzyme may become activated and cut the
DNA wrongly, with potentially harmful consequences.

To explore whether the S. aureus Cas9 could be modified to cleave with
higher fidelity to the intended target, the authors generated a range
of novel Cas9 mutants and tested their ability to discriminate against imperfect matches while retaining high activity at the intended site. They found one such mutant, which distinguished and rejected single base-pair mismatches between gRNA and DNA, regardless of the target, increasing
the fidelity up to 93-fold over the original enzyme. They showed that
the mutation affected part of the recognition domain, the region of the
enzyme that coordinates contacts between the enzyme and the gRNA-DNA
complex. The mutation had the likely effect of weakening those contacts,
thus ensuring that only the strongest pairing -- which would come from
a perfect sequence match -- would trigger enzyme activity.

"Avoidance of off-target cleavage is a crucial challenge for development
of CRISPR for medical interventions, such as correcting genetic diseases
or targeting cancer cells," Gu said. "Our results point the way to
developing potentially safer gene therapy strategies."

========================================================================== Story Source: Materials provided by PLOS. Note: Content may be edited
for style and length.


========================================================================== Journal Reference:
1. Haihua Xie, Xianglian Ge, Fayu Yang, Bang Wang, Shuang Li,
Jinzhi Duan,
Xiujuan Lv, Congsheng Cheng, Zongming Song, Changbao Liu,
Junzhao Zhao, Yu Zhang, Jinyu Wu, Caixia Gao, Jinwei Zhang, Feng
Gu. High-fidelity SaCas9 identified by directional screening
in human cells. PLOS Biology, 2020; 18 (7): e3000747 DOI:
10.1371/journal.pbio.3000747 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200709141619.htm

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