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A team led by researchers at Massachusetts General Hospital (MGH) has overcome major limitations
in cutting and editing DNA through the CRISPR-Cas enzyme and other technologies.
The recent innovation, published in the journal Nature Biotechnology, will simplify and accelerate molecular cloning methods and expand their usefulness
CRISPR-Cas editing has changed researchers' ability to alter DNA — for example, cutting specific DNA sequences in ways that restriction enzymes or proteins isolated from bacteria cannot, and this protein has been used to cut DNA sequences at specific sites for decades
Although the CRISPR-Cas tool can be programmed to locate and cut almost any DNA sequence, a major limitation of its localization is the requirement to first identify short sequences located on the flank of the target, called the proto-spaced adjacent motif (PAM).
As a result, DNA could previously only be cut
on the side of this particular motif.
In this latest study, the team previously designed a variant of CRISPR-Cas9 with almost no PAM, called SpRY, to test its utility as a universal DNA-cutting tool
By designing the SpRY and guide RNA (gRNA) complexes to target more than 130 DNA sequences in laboratory experiments, the scientists were surprised to find that SpRY was PAMless-free in vitro and could efficiently cut DNA
from any sequence programmed by gRNA.
The researchers also showed that their technique can overcome the limitations
of restriction enzymes.
"We demonstrated that SpRY DNA digestion (sprygests) enables DNA cutting in almost any sequence, including a wide range, that could not previously be targeted with restriction enzymes or other CRISPR-Cas proteins," said senior author Benjamin Kleinstver, Ph.
, a research assistant at the Center for Genomic Medicine at Massachusetts General Hospital and an assistant professor
at Harvard Medical School.
This new method allows researchers to select any DNA location in the test tube to cut DNA
The new capabilities offered by SpRYgests will accelerate and reduce the cost of a variety of basic research applications, including research
that may ultimately have clinical impact.
The researchers envision that SpRYgests can be widely used to simplify typical molecular cloning methods, more complex cloning methods, assemble next-generation sequencing libraries, and many others
The study was conducted by postdoctoral fellow Kathleen A.
Led by Dr.
Christie, she vowed never to use restriction enzymes again
Other co-authors include Jimmy A.
Guo, Rachel A.
Silverstein, Roman M.
Doll, Megumu Mabuchi, Hannah E.
Stutzman, Jack-Chung Lin, Marlin-yuan, Russell T.
Walton, Luca Pinello, and G.
This work was supported by the National Institutes of Health and the
National Science Foundation.
Precise DNA cleavage using CRISPR-SpRYgests