Explainer: Basics of CRISPR genome editing

In December 2014, the MIT Technology Review described CRISPR as “the biggest biotech discovery of the century.” This was not an over stretch. CRISPR genome editing or CRISPR/Cas system genome editing is a “search and replace” function for DNA. It could easily disable genes or change their functions by deleting or replacing DNA letters or sequence.

Studies related to the therapeutic applications of CRISPR genome editing are promising. These ranges from producing genetically modified organisms to reengineering the body to cure genetic disorders. Some studies suggest possibility for rendering HIV unable to replicate inside an infected immune cell.

Defining and understanding genome editing

Genome editing or genome editing with engineered nucleases is a specific genetic engineering process that involves using engineered nucleases or molecular scissors to insert, delete, or replace DNA. In therapeutic application, gene editing is tantamount to reengineering the body to correct genetic disorders or create a response to a particular illness by inducing targeted mutations.

A literature review Yoshio Koyanagi et al mentioned that there have been several genome-editing methods developed over the last decade. One method involves using artificial nucleases such as zinc finger nucleases or ZFNs and transcription activator like-effector nucleases or TALENs. These molecularly engineered nucleases are able to induce targeted mutations by inserting, deleting, or editing DNA.

Several applications of ZFNs and TALENs genome editing have produced beneficial results. These include targeted gene modification in plants and gene therapy. However, Koyanagi et al reiterated that both ZFNs and TALENs remain somewhat difficult and time-consuming to design and develop.

In 2013, researchers Jennifer Doudna and Emmanuelle Charpentier discovered another genome editing method that involved using the CRISPR system. Compared to ZFNs and TALENs, CRISPR genome editing is simpler to design and develop.

Other researchers have explored further the properties and application of CRISPR. The emerging discoveries create high hopes for using CRISPR genome editing or CRISPR/Cas system genome editing in different medical and biotechnological applications.

Brief history of CRISPR discovery and research

The year was 1987 when Yoshizumi Ishino and her colleagues at Osaka University in Japan published a study detailing the sequence of a gene called iap found in the gut microbe E. coli. To better understand how this gene worked, they also sequenced some of the DNA surrounding it. They found that near the iap gene is a clustered repeats or identical segments of DNA. However, the function of these repeats was not clear at this time.

Other independent studies also reported these clustered repeats present in other species of bacteria and archaea. These findings reported that each repeat sequences were separated from each other by so-called DNA spacers. These spacers had a unique sequence unlike the repeat sequences.

A study published in 2002 and conducted by researchers Ruud Jansen et al collectively called the repeat sequences and the DNA spacers between them as clustered regularly interspaced short palindromic repeats or CRISPR. Jansen et al also explained that a collection of genes accompanied the CRISPR sequences. They called these genes CRISPR-associated genes or Cas genes.

What was interesting about this Cas genes was that they encoded putative nuclease or helicase protein—or enzymes that that could cut or unwind DNA. Jansen et al were unable to explain why these genes did so. Furthermore, researchers could not explain why these Cas genes always sat next to the CRISPR sequence.

In 2005, three separate studies from separate researchers noticed something odd about CRISPR spacers: They resembled viral DNA and extrachromosomal DNA such as plasmids. The three group of researchers drew several conclusions and suggestions from their studies.

The study of C. Pourcel, G. Salvignol, and G. Vergnaud concluded that the CRISPR structure provides a new and robust tool identification tool for evolutionary studies. Another study by Elena Soria et al concluded that the extrachromosomal elements in CRISPR fail to infect the specific spacer-carrier strain, thus implying a relationship between CRISPR and immunity against targeted DNA. The study of S. Dusko Ehrlich et al also suggested that the spacer elements in CRISPR are the traces of past invasions by extrachromosomal elements. They further hypothesised that CRISPR provide the cell immunity against phage infection and foreign DNA expression by coding an anti-sense RNA.

What is the CRISPR/Cas system? What are its implications?

Further studies about CRISPR confirmed previous hypothesis. As it turned out, CRISPR works together with Cas and crRNA to form the CRISPR/Cas system. This complex is a naturally-occurring defence mechanism found in a wide range of bacteria and archaea. J. A. Doudna and her team described CRISPR as essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea.

It is important to highlight the fact that CRISPR are segments of bacterial or archaea DNA containing short repetition of base sequences. Between repetitions is a short segment of spacer DNA or intergenic spacer acquired from previous exposure to plasmids or virus. The entire CRISPR/Cas system is a complex composed of CRISPR RNAs or crRNAs and CRISPR-associated proteins or Cas proteins. This complex works by degrading the complimentary sequences of invading viral or plasmid DNA.

Doudna et al explained that the CRISPR-mediated immune systems depend on small RNAs for sequence-specific detection and silencing of foreign genetic elements. The systems form part of the greater immune response of prokaryotic. To better illustrate, whenever a plasmid or virus invades a microbe, the microbial cell grabs some genetic material from the invader, cuts open its DNA, and inserts the plasmid or viral genetic material into a spacer.

The result of this foreign DNA insertion is the CRISPR/Cas system. This system or complex represents the encounter or exposure to plasmid and viral genetic materials. As an adaptive immune system, the microbe uses the obtained foreign genetic material to turn Cas enzymes into an targeted immune response mechanism alongside RNA targeting. This works by copying the foreign genetic material from each spacer and turning it into an RNA molecule or crRNA molecule. The Cas enzyme then integrates this crRNA molecule. Whenever the microbe reencounters similar plasmid or virus, the crRNA recognises and latches on plasmid or viral DNA sequence while the Cas enzyme cut the plasmid or viral DNA in two to prevent it from replicating.

In other words, the CRISPR system is a prokaryotic adaptive immune system that confers resistance to foreign genetic elements such as plasmids and virus through RNA-guided genetic silencing. To a certain extent, the entire CRISPR array reflects the history of encounters with invading genetic elements.

Application of CRISPR as genome editing tool

The properties and mechanism of CRISPR/Cas system makes it a probable genome editing tool—specifically for inserting, deleting, or replacing DNA. According to MIT Technology Review article, American biochemist and molecular biologist Jennifer Doudna and French molecular biologist and geneticist Emmanuelle Charpentier have been largely credited for introducing the CRISPR/Cas system as a novel genome editing tool.

Both Doudna and Charpentier collaborated to explore and understand the molecular mechanism of a particular CRISPR/Cas system that uses the specific Cas protein called Cas9 found in the Streptococcus bacteria—the bacteria commonly responsible for strep throat infection or sore throat.

Their study demonstrated that Cas9 could be used to make cuts in DNA sequence desire. Take note that this demonstration involved combining Cas9 with synthetic or lab-developed guide RNA molecule. The RNA molecule could be designed to match a sequence of DNA researchers are targeting to edit.

Nonetheless, Doudna and Charpentier concluded that there is a high potential or exploiting the specific CRISPR/Cas9 system for RNA-programmable genome editing. This means that researchers could design a CRISPR/Cas9 system that could work in virtually any organisms they chose to work.

There are currently other researchers and startup companies working on to understand exploit CRISPR genome editing and CRISPR/Cas system further. Other studies reported that using the system for activating or silencing genes. The CRISPR/Cas system opens possibility for curing genetic disorders and even cancers. It is worth mentioning that the CRISPR/Cas system provides a simple tool for mimicking diseases or demonstrating what happens when a gene is knocked down or mutated.

Furthermore, there are also studies that demonstrated the use of this genome editing tool to rid cells of infections. Some studies have demonstrated the use of CRISPR/Cas system at the germline level to create species with desire traits by targeting specific genes.

The possibilities from CRISPR genome editing are starting to emerge as researchers and even the business community have been pouring their resources to perfect or discover novel applications in the fields of medicine and biotechnology.

Further details of the MIT Technology Review are in the article “Who Owns the Biggest Biotech Discovery of the Century” authored by Antonio Regalado and published in December 2014. Details of the study of Koyanagi et al are in the article “Harnessing the CRISPR/Cas9 System to Disrupt Latent HIV-1 Provirus” published in August 2013 in the journal Scientific Reports.

Further details of the study of Ishino et al are in the article “Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product” published in December 1987 in the Journal of Bacteriology. Details of the study of Jansen et al are in the article “Identification of Genes that are Associated with DNA Repeats in Prokaryotes” published in March 2002 in the journal Molecular Biology.

Further details of the study of Pourcel et al are in the article “CRISPR Elements in Yersinia pestis Acquire New Repeats by Preferential Uptake of Bacteriophage DNA, and Provide Additional Tools for Evolutionary Studies” published in March 2005 in the journal Microbiology. Details of the study of Soria et al are in the article “Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements” published in February 2005 in the Journal of Molecular Evolution. Details of the study of Ehrlich et al are in the article “Clustered Regularly Interspaced Short Palindrome Repeats have Spacers of Extrachromosomal Origin” published in February 2005 in the Journal of Molecular Evolution.

Further details of the study of Doudna et al are in the article “RNA-guided Genetic Silencing Systems in Bacteria and Archaea” published in February 2012 in the journal Nature. Details of the study of Doudna and Charpentier are in the article “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity” published in August 2012 in the journal Science.