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CRISPR 101: What is CRISPR-Cas9?

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CRISPR 101: What is CRISPR-Cas9?

By Kevin Holden, PhD, Head of Synthetic Biology at Synthego

This is the first part in the CRISPR 101 series by Synthego, providing a crash course on CRISPR-Cas9 and its applications in a wide range of life science disciplines.

The relative ease of CRISPR gene editing is revolutionizing how scientists conduct genome engineering. However, researchers new to the technology can be overwhelmed with the process and its potential in the lab. In a recent survey conducted by Synthego, it was found that 87 percent of new CRISPR users are also new to gene editing, demonstrating the tremendous interest in CRISPR’s effectiveness and simplicity as compared to other gene editing methods. The below serves as an introduction to the technology, its development, and current applications in the lab.

History

While CRISPR technology wasn’t explored until 2007, the foundational discoveries leading up to it were uncovered back in 1993. Back then, genomic regions in bacteria known as Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, were first identified. In 2007, researchers discovered that CRISPR functioned as a form of microbial adaptive immunity. Since then, researchers have been working tirelessly to clarify the underlying molecular mechanisms behind CRISPR in Prokaryotes.

By 2012, it was realized that this powerful technology could be adapted for engineering the genomes of microbes, plants, animals and other varieties of cells. Fast forward to today, where CRISPR has truly revolutionized genome engineering. Its adaptable nature allows it to have a wide effect across a broad range of species, and its adoption and implementation in the lab is perpetuating its exponential growth.

What is it?

CRISPR is a form of bacterial immunity that has been adapted by scientists for genome engineering and consists of two components: a specific guide RNA and a non-specific CRISPR-associated endonuclease protein (Cas). The most common CRISPR system utilized for this purpose is derived from Streptococcus pyogenes, known to many of us as the bacteria responsible for Strep throat infections. Specifically, the Cas nuclease from this organism is known as Cas9.

Think of CRISPR-Cas9 as a pair of scissors, following a path similar to when you follow your phone’s GPS. The Cas9 nuclease is directed to a specific DNA sequence target by a guide RNA that is complementary to the DNA target. Once there, the guide RNA binds tightly to the complementary DNA sequence, activating the Cas9 nuclease to specifically cut this DNA strand by causing a double-stranded break. In nature, viruses that infect bacteria insert their genes into a host bacteria in order to replicate. This foreign DNA can get fragmented inside the bacterial cell, and the bacteria store this DNA as CRISPR arrays. Bacteria will express these CRISPR arrays as “guide RNA” fragments and they will duplex with its own Cas9 protein to identify and cut any virus genes of the complementary DNA sequence that may be infecting its genome. Once these genes are cut the cell repairs then in a way that deletes gene function.

The CRISPR-Cas9 system has been successfully adapted by scientists for use in human cells and many other cell types by generating hybrid, single guide RNAs (sgRNA) that target specific sequences of interest. Whereas in nature these sgRNA sequences are limited to foreign viral genes, they can be specifically designed to target theoretically any DNA sequence that contains a common motif. This makes it possible to target millions of sites within a genome. Once at a target, Cas9 cuts the gene or region of interest and is forced to repair it manually or with the help of donor DNA supplied by the researcher. This allows for gene function to be disabled, or for genes to be edited by single bases at time, or for entire genes or regions to be added or removed. The ability to “program” Cas9 to a specific DNA target sequence of a researcher’s choice is one of the truly transformative powers of CRISPR-Cas9. Before the adaptation of this technology, such genomic targeting was practically unheard of.

To learn more about the ins and outs of CRISPR-Cas9, download a free copy of Synthego’s eBook here, and follow the five-part CRISPR 101 series here on Synbiology.

About Synthego

Founded by former SpaceX engineers, Synthego is a leading provider of genome engineering solutions. The company’s flagship product, CRISPRevolution, is a portfolio of synthetic RNA designed for CRISPR genome editing and research. Synthego’s vision is to bring precision and automation to genome engineering, enabling rapid and cost-effective research with consistent results for every scientist.

Headquartered in Silicon Valley, California, Synthego customers include leading institutions in over 32 countries around the world, 8 of the world’s 10 largest biotechnology companies, and 24 of the top 25 global biology universities.

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