Researchers expand target range of CRISPR/Cas Systems

Date:
March 29, 2022
Source:
University of Toronto
Summary:
Researchers have created a genome editing technology that allows
for slight variations in target DNA but retains local specificity,
and which could help realize the potential of CRISPR/Cas-based
gene therapy and pathogen diagnosis.



FULL STORY ========================================================================== Researchers at the University of Toronto have created a genome editing technology that allows for slight variations in target DNA but retains
local specificity, and which could help realize the potential of CRISPR/Cas-based gene therapy and pathogen diagnosis.


========================================================================== Scientists program current CRISPR/Cas systems to recognize and cut precise
DNA sequences, to avoid effects such as snipping the wrong sequence or encouraging unwanted mutations. But that specificity makes it hard for
the systems to identify common variants of a given DNA sequence, which
has partly limited their application.

"A lot of work has gone into making CRISPR/Cas systems more specific,"
said Basil Hubbard, principal investigator on the research who is an
associate professor of pharmacology and toxicology in U of T's Temerty
Faculty of Medicine. "But for certain applications there is also the
need for more flexible targeting in these systems, and our study shows
a possible way to meet that need." The journal Nature Communications
published the findings recently.

CRISPR-Cas systems contain two main molecules: a CRISPR guide-RNA, which contains nucleotide base pairs (adenine, cytosine, guanine and thymine in various combinations) that guide the system to a complementary stretch
of DNA; and a Cas enzyme that cuts the DNA to allow for manipulation of
other genetic code.

The new approach substitutes universal bases for one or more of the four
bases that make up CRISPR guide-RNAs.



==========================================================================
"It functions like an asterisk or wildcard in a digital search, in
areas where we expect variation or don't have data," said Hubbard. "With therapeutics, we can target common variants of the same gene from person
to person, such as single nucleotide polymorphisms. For diagnostics, we
can detect multiple evolved variants of the same pathogen." Experimental CRISPR therapies have shown potential to eliminate genetic disorders,
including sickle cell anemia and muscular dystrophy. But these therapies
do not always work in part due to natural genetic variations among
individuals, according to some studies.

Hubbard said his group's approach could help address this problem, but
that it currently works best in vitro and it would need to run faster
in the cellular environment, perhaps with a re-engineered Cas enzyme.

The technology shows more promise for immediate application in
diagnostics.

Hubbard's lab tested the system's function in eight variants of HIV,
each with differing resistance to current anti-viral drugs. A standard guide-RNA without universal bases detected only three of the eight
variants, while a system with just three universal-base substitutions
found all eight variants.

"There is tremendous diversity among pathogens, especially viruses
and bacteria, and they evolve very quickly," said Hubbard. "The system
works very well to detect that kind of variation, and we think it could
be useful across clinical conditions." Hubbard conceived the idea of
using universal bases while working to make CRISPR/Cas systems more
specific. His lab, then based at the University of Alberta, showed that insertion of synthetic or 'xeno' nucleic acids into guide- RNAs could dramatically reduce off-target gene editing with CRISPR/Cas9.

Off-target effects that cut an incorrect gene could have detrimental
health consequences in a patient, including the development of cancers.

"Our current study gives us more options for tailoring CRISPR
specificity," said Hubbard, who moved his lab to U of T last
fall. "Importantly, the specificity of this system is disrupted only at
the region where we incorporate universal bases and is preserved in other
areas of the sequence, thereby keeping off-target effects to a minimum." Hubbard's lab has filed a patent on the technology and is looking to
partner with a company that specializes in CRISPR diagnostics. He is
hopeful the system will help provide a fast, accurate and cost-effective
way to diagnose several diseases including COVID-19.


========================================================================== Story Source: Materials provided by University_of_Toronto. Original
written by Jim Oldfield.

Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Amanda R. Krysler, Christopher R. Cromwell, Tommy Tu, Juan Jovel,
Basil
P. Hubbard. Guide RNAs containing universal bases enable Cas9/Cas12a
recognition of polymorphic sequences. Nature Communications, 2022;
13 (1) DOI: 10.1038/s41467-022-29202-x ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/03/220329114728.htm

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