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Newly engineered variants of the Cas9 nuclease that significantly reduce off-target activity have been developed. [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]
Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within living organisms. [9] [10] This editing process has a wide variety of applications including basic biological research, development of biotechnological products, and treatment of diseases.
Since the CRISPR-Cas9 was developed from bacterial genome systems, it can be used to target the genetic material in viruses. The use of the enzyme Cas9 can be a solution to many viral infections. Cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information.
See: Guide RNA, CRISPR. Complementary base pairing between the sgRNA and genomic DNA allows targeting of Cas9 or dCas9. A small guide RNA (sgRNA), or gRNA is an RNA with around 20 nucleotides used to direct Cas9 or dCas9 to their targets. gRNAs contain two major regions of importance for CRISPR systems: the scaffold and spacer regions.
It is far less effective at gene correction. Methods of base editing are under development in which a “nuclease-dead” Cas 9 endonuclease or a related enzyme is used for gene targeting while a linked deaminase enzyme makes a targeted base change in the DNA. [69] The most recent refinement of CRISPR-Cas9 is called Prime Editing.
The CRISPR-Cas9 system consists of an enzyme called Cas9 and a special piece of guide RNA (gRNA). Cas9 acts as a pair of ‘molecular scissors’ that can cut the DNA at a specific location in the genome so that genes can be added or removed. The guide RNA has complementary bases to those at the target location, so it binds only there.
Developed to detect off-target mutations from TALEN and CRISPR-Cas9, this technique is based on DNA repair by end joining in DSBs. Once the nuclease is added, it goes on to produce on- and off-target mutations. Along with this there is a bait sequence which also gets cleaved.
CRISPR/Cas9 edits rely on non-homologous end joining (NHEJ) or homology-directed repair (HDR) to fix DNA breaks, while the prime editing system employs DNA mismatch repair. This is an important feature of this technology given that DNA repair mechanisms such as NHEJ and HDR, generate unwanted, random insertions or deletions (INDELs).
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