CRISPR gene editing

CRISPR gene editing (pronounced /ˈkrɪspər/ "crisper") standing for "Clustered Regularly Interspaced Short Palindromic Repeats" is a genetic engineering technique in molecular biology by which the genomes 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.^[1]

The technique is considered highly significant in biotechnology and medicine as it enables editing genomes in vivo very precisely, cheaply, and easily. 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.^[2][3] The third researcher group that shared the Kavli Prize for the same discovery,^[4] led by Virginijus Šikšnys, was not awarded the Nobel prize.^[5][6][7]

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.^[1] 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.^[1] 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 from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways 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.

While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proven 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 became possible. 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.^[8] The ease with which researchers can insert Cas9 and template RNA in order to silence or cause point mutations at specific loci has proven to be 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.^[9]

CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing^[10] was the AAAS's choice for Breakthrough of the Year in 2015.^[11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos.^[12] In 2023, the first drug making use of CRISPR gene editing, Casgevy, was approved for use in the United Kingdom to cure sickle-cell disease and beta thalassemia.^[13][14] Casgevy was approved for use in the United States on December 8, 2023, by the Food and Drug Administration.^[15]