Jan. 12 — One 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 own DNA.

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 cell.

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 (Umeå 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 multiple genes.

The method is improving rapidly and is expected to one day have applications in basic research, drug development, agriculture, and the clinical treatments of 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 molecular level.

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.”

The entire article can be found here.

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Source: Aaron Dubrow, TACC