CRISPR gene editing

CRISPR gene editing (pronounced "crisper") is a genetic engineering technique in molecular biology by which the

genome

s of living organisms may be modified. It is based on a simplified version of the bacterial

CRISPR

-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added ''in vivo'' (in living organisms).

The technique is considered highly significant in biotechnology and medicine as it allows for the genomes to be edited ''in vivo'' with extremely high precision, cheaply, and with ease. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also has possibilities in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer. However, its use in human germline genetic modification is highly controversial. The development of the technique earned

Jennifer Doudna

and

Emmanuelle Charpentier

the Nobel Prize in Chemistry in 2020. The third researcher group that shared the Kavli Prize for the same discovery, led by Virginijus Šikšnys, was not awarded the Nobel prize.

Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of

DNA

to introduce the modification by one of two methods. Knock-in mutations, facilitated via homology directed repair (HDR), is the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted

DNA damage and repair

. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template. This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result in the repair of the double-stranded break by means of non-homologous end joining (NHEJ). NHEJ can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption. Because of this, the precision of genome editing is a great concern. Genomic editing leads to irreversible changes to the genome.

While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proved to be inefficient and impractical to implement on a large scale. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. Cas9 derived from the bacterial species ''

Streptococcus pyogenes

'' has facilitated targeted genomic modification in eukaryotic cells by allowing for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands. The ease with which researchers can insert Cas9 and template RNA in order to silence or cause

point mutation

s at specific loci has proved invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease have been developed that significantly reduce off-target activity.

CRISPR-Cas9 genome editing techniques have many potential applications, including in medicine and agriculture. The use of the CRISPR-Cas9-gRNA complex for genome editing was the AAAS's choice for Breakthrough of the Year in 2015. Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos.

History

Other methods

In the early 2000s, German researchers began developing zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. ZFNs has a higher precision and the advantage of being smaller than Cas9, but ZFNs are not as commonly used as CRISPR-based methods. Sangamo provides ZFNs via industry and academic partnerships but holds the modules, expertise—and patents—for making them. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the design and creation of a custom protein for each targeted DNA sequence, which is a much more difficult and time-consuming process than that of designing guide RNAs. CRISPRs are much easier to design because the process requires synthesizing only a short RNA sequence, a procedure that is already widely used for many other molecular biology techniques (e.g. creating oligonucleotide primers).

Whereas methods such as RNA interference (RNAi) do not fully suppress gene function, CRISPR, ZFNs, and TALENs provide full irreversible gene knockout. CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low.

Discovery

In 2012

Jennifer Doudna

and

Emmanuelle Charpentier

published their finding that CRISPR-Cas9 could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the history of biology

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