CRISPR-Cas9History of Gene scissor for Genome Editing Technology F irst-generation gene editing technique ( Zinc Finger Nucleases, ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequenc and this enables zinc-finger nucleases to target unique sequences within complex genomes. Second-generation gene editing technique ( Transcription activator-like effector nuclease , TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations Third -generation gene editing technique ( Clustered Regular Interspaced Short Palindromic RepeatR location as a new spacer. By encrypting each specific spacer sequence, the CRISPR location proceeds to generate transcription and mature CRISPR RNAs.Discovery of CRISTR-Cas9 CRISPRs were first identified in E. coli in 1987 by a Japanese scientist, Yoshizumi Ishino , and his team, who accidentally cloned an unusual series of repeated sequences interspersed with spacer sequences while analyzing a gene responsible for the conversion of alkaline phosphatase. In 1993 , researchers led by J.D. van Embden in the Netherlands discovered that different strains of Mycobacterium tuberculosis had different spacer sequences between the DNA repeats. Subsequently, these sequences were identified in several other bacterial and archaeal genomes. Researchers Francisco Mojica and Ruud Jansen were the first to refer to them as CRISPRs.CRISPR-CAS9 (Clustered Regular Interspaced Short Palindromic Repeats ) is a natural system used by bacteria to defend themselves against viruses. Tracr RNA (trans-activatinNA), which is complementary to the target segment of the viral genome. The snipped DNA fragment may then be stored between the palindromic CRISPR sequences to retain a genetic memory for disabling future infections from the same viral strain.Invading DNA (Foreign DNA from a virus invades the cell) Invading DNA is incorporated into CRISPR Array (The exact mechanism remains unknown) Pre-crRNA Transcription (The cell constitutively transcribes a repeat/spacer group into pre-crRNA)Guide RNA Formation (Constitutively expressed transactivating RNA base pairs with the CRISPR repeat sequences on the pre-crRNA) Cas9 Activation (inactive Cas9 protein binds to the guide RNA and becomes activated) Target Binding (The activated guide RNA/Cas9 complex blinds with the target DNA) Target cleavage (The Cas9 protein cleaves the invading DNA and inactivates it)Application of CRISTR-Cas9 in the crop improvement 1)Safety Improvement Gliadin (-) wheat Here, we show that CRISPR/Cas9 technology can be used tol 16.4 (2018): 902-910. HOW WHY WHATApplication of CRISTR-Cas9 in the crop improvement 2)Quality Improvement reducing GPC and improving ECQ of rice ECQ is determined by three major endosperm components: starch (80%–90%), protein (7%–10%), and lipids (about 1%) In fact, rice grain protein content (GPC) has a negative effect on chalkiness and ECQ and leads to inferior palatability [1,5,6]. In breeding practice, cultivars with satisfactory ECQ are required to have relatively low (generally 7%) GPC [7]. Managing GPC is essential for improving rice quality, in particular ECQ. We modified the coeliac disease‐causing α‐gliadin gene array using CRISPR/Cas9 technology to obtain nontransgenic , low‐gluten wheat lines. Wang, Shiyu , et al. Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system. The Crop Journal (2020). HOW WHY WHATApplication of CRISTR-Cas9 in the crop improvement 3)Quantity Improvement Development of broad virus resistance hod for the breeding of virus‐resistant crops. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/ subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Chandrasekaran, Jeyabharathy , et al. Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology. Molecular plant pathology 17.7 (2016): 1140-1153. WHAT HOW WHY2020 Nobel chemistry The two discovered what they called genetic scissors. Dr. Emmanuelle Charpentier discovered previously unknown tracrRNA in his study of one of the most damaging bacteria to mankind, called Stretococcus pyogenes. In further research, she used genetic scissors to cut the bacteria into pieces of DNA and disarmed them. Dr. Sharfantier published a related study in 2011 and began working with Jennifer Doudna , a biochemist with extensive knowledge and experience in RNA. They succeeded in reproducing theow}
