Things You Should Know About CRISPR Gene-Editing Tool

CRISPR gene editing technology is driving every aspect of biotechnology including molecular biology, genetics, oncology, immunology, agricultural and industrial biotechnology, and even food technology. Since its discovery, new companies have been founded to deliver advantages of CRISPR to various sectors and many of the existing gene editing companies are beginning to harness the power of CRISPR.

What is CRISPR?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR is a set of DNA sequences found in the genomes of prokaryotes like Archaea and bacteria. CRISPR sequences are characterized by clusters of identical repeats interspaced with non-identical segments called spacers. These prokaryotic DNA elements are derived from viruses when they attack the prokaryote. Subsequently, these DNA sequences are involved in adaptive immunity against invasive genetic elements including viruses and plasmids. The CRISPR segments along with certain enzymes constitute a basis of a powerful gene editing system. Thus, in the modern biological fields, the term CRISPR denote more of a genome-editing tool than just the gene sequences coding for immunity in bacteria.

Crispr Stand For Clustered Regularly Interspaced Short Palindromic Repeats

Discovery – When and Who Discovered CRISPR?

CRISPR repeats were at first discovered by accident in 1987. It was during the study of the IAP gene responsible for alkaline phosphatase isozyme conversion in E. coli. The research was conducted by Yoshizumi Ishino and group from Osaka University and they discovered a peculiar repeat sequence whose function was unknown at the time. In another independent study of Mycobacterium tuberculosis, researchers in the Netherlands discovered a diversity of cluster of direct repeats (DRs) among different strains of M. tuberculosis. This polymorphic feature of DRs was found to be useful in strain typing and is being used to date. Around the same time, Francisco Mojica and his group in the University of Alicante in Spain observed CRISPR repeats in Haloferax and Haloarcula species of Archaea. However, the role of this clustered palindromic repeats remained mysterious for years.

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Then in 2002, Ruud Jansen and his team coined the term CRISPR and also reported a group of genes dispersed in a cluster in the immediate proximity of CRISPR loci. These genes were termed as CRISPR-associated (Cas) genes. Thus, the first four Cas genes coding for Cas1-4 proteins were discovered. Followed by this, many kinds of research were conducted on CRISPR in various species of bacteria throughout the world. Eventually, scientists reported that the CRISPR spacers were derived from viruses—invading phage DNA and extrachromosomal DNA such as plasmids—when they infected the bacterial cell. This let the scientists uncover the role of the CRISPR/Cas system in adaptive immunity in the prokaryotes.

What is CRISPR-Cas9?

The true CRISPR revolution began when the research teams from the US and Europe led by Jennifer Doudna and Emmanuelle Charpentier revealed how this bacterial defense system could be turned into a phenomenal genome-editing tool. The two scientists studied the CRISPR system in Streptococcus pyogenes, a gram-positive pathogenic bacterium with Cas9 protein involved in its adaptive immune system.

The Cas9 (CRISPR associated protein 9) is a DNA endonuclease enzyme associated with CRISPR adaptive immunity in S. pyogenes. Cas9 is an RNA-guided endonuclease and thus can cleave nearly any sequence of DNA complementary to the guide RNA.

How does CRISPR-Cas9 work?

How Crispr works

Credit – MRS Bulletin

The Cas9 endonuclease system uses two small RNA molecules called CRISPR RNA (crRNA) and two trans-activating CRISPR RNA (tracrRNA). In naturally occurring CRISPR-Cas system, once the crRNA-tracrRNA-Cas9 effector complex is ready, Cas9 looks for a specific protospacer adjacent motif ‘PAM’ in the phage or plasmid DNA sequence. This PAM sequence is present only in the phase or viral DNA, in close proximity with protospacer itself, but it is not incorporated in the CRISPR sequence in bacteria. This way the CRISPR-Cas9 system recognizes new protospacer and identifies invaders upon infection, thus avoiding cleavage of its own CRISPR sequence and providing immunity against viruses. This has thus become the basis of CRISPR/Cas9 genome editing.

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How Crispr works

Doudna and Charpentier re-engineered the Cas9 endonuclease by fusing (base-pairing) crRNA with tracrRNA forming a two-RNA structure that directs the Cas9 protein to introduce double-stranded (ds) break in target DNA. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. That is, by manipulating the nucleotide sequence of the guide RNA, the Cas9 system could be programmed to target any dsDNA sequence for cleavage. In other researches scientists have modified the CRISPR system to make programmable transcription factors that allow to target and activate or silence specific genes, therefore, making it possible for sequence-specific control of gene expression on a genome-wide scale.

In Conclusion

CRISPR is, therefore, revolutionizing modern biotechnology and molecular biology. Studies have shown that the CRISPR system is present in nearly 40% of bacteria and 90% of archaea. But with the applications of biotechnology this natural feature of prokaryotic organisms is becoming a much larger tool to disseminate its applications of genome editing to wide range of organisms including baker’s yeast (Saccharomyces cerevisiae), Candida albicans, Drosophila melanogaster, ants, mosquitoes, nematodes, plants, mice, monkeys, et cetera and even human embryos. The CRISPR/Cas9 has become as much consequential as the zinc finger nucleases and TALEN proteins. While many pre-existing genome editing companies have adopted CRISPR, a large number of CRISPR startups have been established throughout the globe. From agriculture and food technology to pharmaceuticals and therapeutics, CRISPR is truly transforming the realm of biotechnology.


CRISPR Resources