CRISPR-Cas is a gene editing technology that is revolutionizing the biotech industry. In 2012, two researchers were performing basic research when they inadvertently discovered a gene editing technology that forever changes how scientists edit genes.
Emmanuelle Charpentier and Jennifer A. Doudna were trying to gain a better understanding of the system that bacteria use to defend themselves against viruses. The researchers noticed that bacterial systems could slice out specific sections of DNA with incredible precision. The team then realized that, with the correct programming, the system could also replace a specific section with new DNA – in essence, the bacteria were performing gene editing on their own DNA in a better way than scientists were doing it in the laboratories.
Feng Zhang, PhD, of the Broad Institute of MIT & Harvard, George Church of Harvard University, was just ahead of Doudna’s group, using his patented CRISPR-Cas system along with other methodologies to investigate genetic and epigenetic mechanisms and their roles in underlying diseases. Once called the “Midas of Methods” by The Scientist, Church now makes these tools widely available.
Like all organisms, the bacteria that Zhang and Doudna's team studied had to adapt continuously to changes in their environment in order to survive. The prokaryotics, bacteria and archaea, use horizontal gene transfer to acquire new traits quickly. This new DNA can cause damage, however, so it is essential that the organism remove imported DNA and protect against damaging DNA elements. Balance between DNA uptake and DNA degradation is also important.
The History of CRISPR
In the 1980s, scientists noticed an intriguing pattern in some bacterial genomes, where a DNA sequence would be repeated several times with unique sequences in between the repeated sections. They called these sequences “clustered regularly interspaced short palindromic repeats,” or CRISPRs. Scientists discovered that the unique sequences matched the DNA of viruses, specifically viruses with an affinity for bacteria.
Prokaryotics keep chunks of dangerous viruses around to help them recognize and defend against these viruses in subsequent attacks. Virus DNA fills various CRISPR regions to become a sort of “molecular most-wanted gallery” that represents all the viruses the microbe has encountered. The microbe then uses this DNA to turn CRISPR-associated proteins (Cas) into a precision weapon by copying the genetic material of a particular CRISPR region into an RNA molecule.
CRISPR and Cas Enzymes
CRISPR-associated proteins are a set of enzymes that can snip precise bits of DNA and slash invading viruses. The genes that encode for Cas are usually conveniently located near CRISPR sequences. Cas enzymes take up and carry the RNA molecules as they drift around the cell. If Cas enzymes and the RNA molecules they cradle encounter genetic material that matches CRISPR RNA, the RNA molecules grasp onto the invader tightly while the Cas enzymes chop the viral DNA in two to prevent it from replicating.
In short, Cas is an enzyme that snips DNA while CRISPR is a collection of DNA sequences that tell the Cas enzymes precisely where to snip. Scientists provide Cas with the right sequence, known as a guide RNA, to cut and paste bits of DNA sequence into any section of the genome they desire.
There are a number of Cas enzymes but CRISPR uses Cas9, derived from Streptococcus pyogenes that is best known as the cause of strep throat. Other enzymes might make a cut every time they encounter a particular sequence, potentially dicing up the entire genome, but Cas9 can recognize very long sequences so scientists can tailor it to target a specific gene. They can even repair a faulty gene by cutting it out with CRISPR and injecting a normal copy into the cell. Scientists design a target sequence and obtain the guide RNA to match.
Pros and Cons of Other Gene Editing Tools
CRISPR-Cas9 joins the other families of engineered nucleases, including Zinc finger nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). ZFNs and TALENs use artificial restriction enzymes to take advantage of the action of endogenous DNA repair machinery. ZFNs and TALENs can recognize longer DNA sequences and, theoretically, they have better specificity than CRISPR/Cas9.
The downsides to ZFN and TALENs, however, are that scientists have to create new custom-designed ZFN or TALEN protein each time they want to edit a gene sequence and that they frequently must create several variations before they find one that works. CRISPR-Ca9 technology is more efficient in that it allows researchers to create an RNA guide sequence easily.
CRISPR as Disruptive Technology
CRISPR-Cas9 gives scientists a better way to edit genes for biotechnology research. Knockout mice are the workhorses of biomedical research, for example, but establishing new lines of genetically altered mice can take up to a year and three generations with traditional methods using embryonic stem cells. Using CRISPR, on the other hand, produces knockout mice in just one generation. Furthermore, researchers can alter several genes at once.
Scientists used to spend months, or even years, attempting to rewrite an organism’s DNA. Using CRISPR, now researchers just spend days performing the re-writes. The CRISPR-Cas9 system also allows scientists to perform precision gene editing, especially those working in immunology.
Companies now compete to develop innovating cell therapies and immunotherapies using gene-editing technology. Novartis intends to utilize CRISPR/Cas9 in its immunotherapy program that uses chimeric antigen receptor T-cells (CAR-T) therapies, for example, to eradicate leukemia and lymphoma. Cellectis uses its flagship TALEN™ to create a new generation of immunotherapies. Sangamo uses Zinc finger for human therapeutics, plant agriculture, research reagents, cell engineering applications for protein pharmaceutical manufacturing, and transgenic animal production. Juno Therapeutics has partnered with Editas to expand Juno’s repertoire of cell therapies, which already show amazing promise in patients with blood-borne cancers, to also treat solid tumors. Johnson & Johnson are also using CRISPR technologies to develop new immunotherapies.
CRISPR is changing the landscape of biomedical research with inexpensive, quick and easy-to-use gene editing methods. Labs around the world are switching outdated gene editing technology with CRISPR-Cas9 in hopes of eliminating disease, creating hardier plants, and wiping out pathogens and more.
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