CRISPR Gene Editing

CRISPR gene editing (pronounced "crisper") 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 (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 mutations 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 of Crispr Gene Editing

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.

Patents and commercialization

As of November 2013, SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.  By 2015, Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.

As of December 2014, patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools.  As companies ramped up financing, doubts as to whether CRISPR could be quickly monetized were raised.  In February 2017 the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.  Shortly after, University of California filed an appeal of this ruling.

Recent events

In March 2017, the European Patent Office (EPO) announced its intention to allow claims for editing all types of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna, and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.  As of August 2017 the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.

In July 2018, the ECJ ruled that gene editing for plants was a sub-category of GMO foods and therefore that the CRISPR technique would henceforth be regulated in the European Union by their rules and regulations for GMOs.

In February 2020, a US trial safely showed CRISPR gene editing on three cancer patients.

In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field.  They made history as the first two women to share this award without a male contributor.

In June 2021, the first, small clinical trial of intravenous CRISPR gene editing in humans concludes with promising results.

             https://en.wikipedia.org/wiki/CRISPR_gene_editing