The Year in CRISPRs - 20

The Year in CRISPRs * CRISPR Triple Checks Its Target before Cutting

In the Nature article, the investigators
observed that once Cas9 binds to a region of
DNA, it performs an additional check before
two distal sections of the Cas9 protein complex
come together, like the blades of a scissors, to
precisely align the active sites that cut doublestranded DNA.
"We found that RNA-guided Cas9 can bind
some off-target DNA sequences, which differ
from the correct target by just a few mutations,
very tightly. Surprisingly, though, the region of
Cas9 that does the cutting is inhibited because
of the imperfect match. But when the correctly
matching DNA is located, Cas9 undergoes a large

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| GENengnews.com

structural change that releases this inhibition and
triggers DNA cutting," noted the lead author of
the Nature paper Samuel Sternberg, Ph.D., former
graduate student in Dr. Doudna's laboratory.
"We think that this structural change is the
last checkpoint, or proofreading stage, of the
DNA targeting reaction," Dr. Sternberg continued.
"First, Cas9 recognizes a short DNA segment next
to the target-the PAM-then the target DNA is
matched up with the guide RNA via Watson-Crick
base-pairing. Finally, when a perfect match is
identified, the last part of the protein swings
into place to enable cutting and initiate
genome editing."

When asked about the impact of the current
researcher, Knight responded that the data
"suggests that you have more than one checkpoint
to ensure correct Cas9 binding. There's not just
sequence regulation, there is also temporal regulation: it has to engage with the DNA and park long
enough that it can actually rearrange and cut."
The discoveries from the Berkeley teams
should allow for greater insight into the
molecular mechanisms that lead to off-target
events that can occur during genome editing
applications-an critical area for researches to
grasp an understanding of should they hope to
use CRISPR-Cas9 in a wider clinical capacity. n


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The Year in CRISPRs

Table of Contents for the Digital Edition of The Year in CRISPRs

Contents
The Year in CRISPRs - 1
The Year in CRISPRs - 2
The Year in CRISPRs - 3
The Year in CRISPRs - Contents
The Year in CRISPRs - 5
The Year in CRISPRs - 6
The Year in CRISPRs - 7
The Year in CRISPRs - 8
The Year in CRISPRs - 9
The Year in CRISPRs - 10
The Year in CRISPRs - 11
The Year in CRISPRs - 12
The Year in CRISPRs - 13
The Year in CRISPRs - 14
The Year in CRISPRs - 15
The Year in CRISPRs - 16
The Year in CRISPRs - 17
The Year in CRISPRs - 18
The Year in CRISPRs - 19
The Year in CRISPRs - 20
The Year in CRISPRs - 21
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