UNT's Jin Liu Catches CRISPR in
January 16, 2017
University of North Texas researchers use supercomputers to create first
all-atom simulation of genome editing in action
of the most talked about biological breakthroughs in the past decade was
the discovery of the genome editing tool CRISPR/Cas9, which can alter
DNA and potentially remove the root causes of many hereditary diseases.
Originally found as part of the immune system of the Streptococcus
pyogenes bacteria, CRISPR associated protein 9 (CAS9), in its native
state, recognizes foreign DNA sequences and disables them.
In bacteria, the system is used to target foreign viral DNA from
bacteriophages - DNA that it has already recognized as an enemy through
its evolutionary history and has incorporated a record of it into its
CRISPR (Clustered regularly interspaced short palindromic repeats,
pronounced "crisper") represent segments of DNA that contain short
repetitions of base sequences followed by short segments of "spacer DNA"
derived from previous exposures to foreign DNA. The complex consists of
proteins that unravel DNA, others that cut the double helix at a
specific location, and a guide RNA that can recognize enemy DNA in the
A visual description of the CRISPR/Cas9
research explored by UNT researchers using TACC supercomputers.
Simulations found that Cas9 produces staggered, "sticky" ends, which may
make them easier to manipulate for future use.
Researchers studying this ancient
immune system realized that, by changing the sequence of the guide RNA
to match a given target, it could be used to cut not just viral DNA, but
any DNA sequence at a precisely chosen location. Furthermore, new
sections of DNA could be introduced to join to the newly cut sections.
The method was first conceived and developed by Jennifer Doudna
(University of California, Berkeley) and Emmanuelle Charpentier (UmeŚ
University) and has been used in cultured cells -- including STEM cells
-- and in fertilized eggs to create transgenic animals with targeted
mutations that help study genetic functions. CRISPR/Cas9 can affect many
genes at once, allowing for the treatment of diseases that involve the
interaction of many genes.
The method is improving rapidly and is expected to one day have
applications in basic research, drug development, agriculture, and
treating human patients with genetic diseases.
However, creating targeted CRISPR/Cas9 mutations is currently expensive
and time-consuming, particularly for large-scale studies. The process is
also error prone, limiting its widespread use. These problems stem, in
part, from a lack of full understanding of how CRISPR/Cas9 works at the
In November, a research team from the University of North Texas (UNT)
led by Jin Liu used the Maverick supercomputer at the Texas Advanced
Computing Center (TACC) to perform the first all-atom molecular dynamics
simulations of Cas9-catalyzed DNA cleavage in action.
The simulations, reported in Nature Scientific Reports, shed light on
the process of Cas9 genome editing. They also helped resolve
controversies about specific aspects of the cutting: such as where
precisely the edits occur and whether Cas9 generates blunt-ended or
staggered-ended breaks with overhangs in the DNA.
"Right now there are quite a few problems in how we use this in the
therapeutic applications. The specificity and efficiency of the enzyme
are not high," Liu said. "It is also difficult to deliver the enzyme to
the position of the gene editing. To solve these problems, first, we
need to know how this enzyme works. Our research is providing the
foundation for the understanding of the mechanism of Cas9."
Scientists had previously determined the structure of Cas9 in its
inactive state through x-ray crystallography, but the gene editing
process happens so fast that no experimental technique could capture its
state changes and determine exactly how it works.
Using molecular dynamics simulations -- a method of studying the
dynamics of molecules by simulating the interactions of a large numbers
of atoms over a fixed time -- Liu and co-author Zhicheng Zuo, also at
UNT, were able to observe the changes in the Cas9 enzyme in femtosecond
resolution and capture the transition to the system's active state.
The simulations included Mg2+ ions, which are believed to induce the
conformation changes in Cas9 so it can function. The researchers
hypothesized that the Mg2+ ions induce the conformational change to an
active state by facilitating the proximity of Cas9 active sites and DNA.
The simulations Liu and Zuo ran on Maverick encompassed 280,000
interacting atoms. The most challenging part, according to Liu, was
effectively sampling conformation spaces to locate the active state.
"We tried our best to enhance the reliability of our simulation
results," Liu said. "We did several long 200 and 300 nanosecond
trajectories, and we also did several short trajectories of 60
nanoseconds, to ensure we had consistent results. For each
configuration, we performed at least six parallel simulations."
In addition to studying how the CRISPR/Cas9 complex changes into its
active state, the simulations shed a light on where it snipped the DNA.
"Normally people believe it is cut at one position, but there is no
clear evidence showing that," said Liu, assistant professor of
Pharmaceutical Sciences at UNT. "Using our computational methods, we
determined the cutting occurs at another position, which could mean that
this enzyme could cut DNA in a more controllable way. So, by doing this
work, we open the door for designing a more efficient enzyme that could
cut DNA in a more specific way."
Their final contribution answered whether genome editing generated blunt
ends or staggered ends -- an unresolved question with practical
CRISPR/Cas9 cuts both halves of a double-stranded DNA. If it cuts at the
same position on both strands it would generate blunt ends; if it cuts
at a slightly different position, it would create staggered ends. Their
simulations suggested CRISPR/CAS9 creates staggered ends.
"This will be quite critical for gene editing because staggered-ended
DNA is more controllable than the blunt-ended DNA," she said.
Iok-Hou Pang, chair of Pharmaceutical Sciences at UNT, uses CRISPR/Cas9
to do experimental eye research in his lab. For both his current and
future research he sees the value of a clearer understanding of the
lab has been using the CRISPR/Cas9 gene editing system to knockdown a
novel protein in cultured mouse optic nerve astroglial cells," said
Pang. "Dr. Liu's work will help us understand the molecular interaction
between the enzyme system and its substrate and will eventually help to
improve its efficiency, which is tremendously useful for this wonderful
In 2015, under the auspices of the National Academies of Science, global
scientists declared a moratorium on using CRISPR/Cas9 to edit the human
genome in ways that could be inherited (though there are indications
that researchers have already broken this injunction). Too little, they
said, was known about the process and the long-term effects of doing so.
But research like Liu's could one day play a role in fine-tuning these
tools and making them feasible for human use.
"We're trying to understand the mechanism of the genome editing enzyme
that could have great potential toward future pharmaceutical and
therapeutic applications and that might get us closer to a position
where this technology could be used on human diseases," Liu said
"Without TACC resources this research would be impossible to perform."