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CRISPR gene editing is a revolutionary technology that allows for precise, targeted modifications to the DNA of living organisms. Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is the most commonly used system, that allows "cutting" of DNA at specific locations and either delete, modify, or insert genetic material.
Cas9 has been used often as a genome-editing tool. Cas9 has been used in recent developments in preventing viruses from manipulating hosts' DNA. 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.
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]
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.
Many approaches have since been developed to improve the efficiency of mutagenesis. A large number of methods are available to effect site-directed mutagenesis, [ 12 ] although most of them have rarely been used in laboratories since the early 2000s, as newer techniques allow for simpler and easier ways of introducing site-specific mutation ...
The CRISPR/Cas9 system is based on an adaptive immune system of prokaryotic organisms, and its use for genome editing was first proposed and developed in collaboration between Jennifer Doudna (University of California, Berkeley) and Emmanuelle Charpentier (Max Planck Institute for Infection Biology, Germany). [1]
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.
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.
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