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GENE CORRECTION.pptx
- 1. PRESENTED BY S.R. BHARATHKUMAAR, I M.Sc BIOTECHNOLOGY 2019-2021 , BHARATHIAR UNIVERSITY.
- 2. Gene corrections is a technology that gives us the tools for both repairing and mutating DNA, for discovering gene functions and for engineering new genetic variants.
- 3. Gene correction technology using tailor made nucleases such as Mega nucleases (MNs) Zinc finger nucleases (ZFNs) TAL effector nucleases (TALENs) CRISPR/Cas9
- 4. Mega nucleases “Molecular DNA scissors” that can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Zinc-finger nucleases Zinc-finger nucleases are powerful gene modification tools The ultimate maneuver in gene therapy is to replace a defective gene with a normal allele at its natural chromosomal location. In principle this has several advantages over providing a complete therapeutic gene e.g., with a viral vector
- 5. TAL Effector Nucleases (TALENs) 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 cleavage domain ( a nuclease which cuts DNA strands). Transcription activator like effectors (TALEs) can be engineered to bind to particularly any desired DNA sequence, so when combined with a nuclease, DNA cut at specific locations The enzymes introduced into cells for gene correction
- 6. CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats Use to understanding, characterizing and controlling DNA; Based on bacterial immune system; single solution to many problems CRISPR associated system protein 9; An RNA-guided DNA endonuclease enzyme associated with CRISPR Cas 9 protein is responsible for locating and cleaving target DNA, both in natural and in artificial CRISPR/Cas systems A prominent tool in the field of genome editing Advance in investigation, prevention, treatment of diseases, to increase cropyield and to understand the function of the gene
- 7. TARGET DNA RECOGNITI ON DNA CLEAVAGE CONSTRUCTION NUCLEASE TARGET SITE RECOGNI TION SIZE MN Homing endonuclease Homing endonuclease Randomized mutilation at the DNA recognition residues and screening of combinatorial library 22 bp ZFN Zinc finger domains FokI nuclease domain Assembly of 3-4 zinc finger domains and screening for context dependent binding specificity (9 or 12 bp) 9 2 TALE N RVD (repeat variable diresidue) repeats FokI nuclease domain Assembly of 8-31 RVD repeats (8–31 bp) 9 2 CRISP R/Cas9 CRISPR RNA (crRNA) or guide RNA (gRNA) Cas9 Oligonucleotide synthesis of gRNA and molecular cloning (or RNA synthesis) 20 bp + “NGG” (*1) Table 1. Various engineered nucleases
- 9. Nucleases Cell type Disease Cause of disease Correction ZFN CD4+ T cells AIDS HIV infection Disruption of CCR5/ ZFN CD34+ HSCs AIDS HIV infection Disruption of CCR5/ Disruption CRISPR CD4+ T cells or 293T cells AIDS HIV infection Disruption of HIV LTR or excision of HIV provius TALEN iPSCs Hepatitis C HCV infection Disruption of APOB TALEN Fused dermal fibroblasts Kearns- Sayre syndrome 5 kb deletion in mtDNA Ellimination of mutant mtDNA
- 10. Nucleases Cell type Disease Cause of disease Correction ZFN Myoblasts Duchenne muscular dystrophy Dystrophin Dexon 51, 51–53, 51–60 (1 + 3n) bp frameshift at exon 50 Frameshift TALEN Immortalize d myoblasts Duchenne muscular dystrophy Dystrophin Dexon 48– 50 (2 + 3n) bp frameshift at exon 51 ZFN CD4+ T cells X-linked SCID X-linked SCID 1 bp frameshift at exon 5
- 11. Nucleases Cell type Disease Cause of disease Correction ZFN Immortalize d B lymphocytes X-linked SCID IL2Rc truncation in or after exon 5 Knock-in IL2Rc cDNA (exon 5–8) Knock-in MN Immortalize d myoblasts Duchenne muscular dystrophy Dystrophin Dexon 45– 52 Knock-in exon 45–52 ZFN Mouse liver containing hF9 Hemophilia B F9 Y155 stop Knock-in hF9 cDNA (exon 2–8) at intron 1 ZFN iPSCs Down syndrome Trisomy 21 Knock-in Xist to inactivate the trisomic chr 21
- 12. Nucleases Cell type Disease Cause of disease Correction ZFN iPSCs Parkinson’s disease a-synuclein Thr53Ala (A > G) ZFN iPSCs Sickle-cell anemia b-globin Val6Glu (T > A) Substitutio n TALEN iPSCs Metabolic liver disease a1- antitrypsin Lys342Glu (A > G) TALEN iPSCs Epidermolys is bullosa COL7A1 Stop613Arg (T > C)
- 17. THANK YOU