gene editing tools of biotechnology.pptx

Gene editing tools are used to alter the genome at specific locations. They work by directing enzymes to a target DNA sequence,where they can cut the DNA and be used to edit it.

1. GENE EDITING TOOLS By Tanishka Bhadauriya under the guidance of Tajendra sir Bsc III year 21310061

2. INTRODUCTION TO GENE EDITING AND ITS TOOLS Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerase. Today we have more accurate techniques such as CRISPR/ Cas and TALENs.

3. GENOME EDITING: THREE STEPS Target the locus Delete or insert a gene Repair the DNA strand

4. GENE EDITING TOOLS

5. What is CRISPR ?

6. CRISPR • CRISPR is a family of DNA sequences in bacteria. • The sequence contains snippets of DNA from viruses that have attacked the bacterium. • These snippets are used by the bacterium to detect and destroy DNA from further attacks by similar viruses. • Small clusters of Cas ( CRISPR – associated system) genes are located next to CRISPR sequences. • The CRISPR/ cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present with plasmids and phages that provides a form of acquired immunit y.

7. THREE TYPES OF CRISPR SYSTEMS

8. Cas – 9 (CRISPR associated protein 9) Is an RNA guided DNA endonucleases enzyme Associated with CRISPR Plays an important role in adaptive immunity system , found in bacteria Streptococcus Pyogenes . Involved in type || CRISPR mechanism.

9. BIOLOGICAL STRUCTURE OF CAS 9 Cas 9 protein has six domains- • REC | responsible for binding guide RNA. • REC || not yet well understood • Bridge helix (arginine rich) is crucial for initiating cleavage activity upon binding of target DNA. • PAM interacting domain confers PAM specificity ; responsible for initiating binding to target DNA. • HNH and RuvC domains are nuclear domains that cut single stranded DNA..They are highly homologous to HNH and RuvC domains found in other proteins.

10. BIOLOGICAL MECHANISM OF ACTION OF CAS 9

11. APPLICATIONS Gene silencing DNA free CRISPR cas 9 gene editing Homology directed repair ( HDR) Transient gene silencing of transcriptional repression ( CRISPRi ) Transient activation of endogenous genes Embryonic stem cell and transgenic animals Pooled genome scale knockout screening

12. TALENs

13. DISCOVERY OF TALENS Transcription Activator Like Effectors (TAL) Discovered in Xanthomonas Injected into plant cells via the type lll secretion systems. Bind to promoter sequence of genes which facilitate bacteria infection Specific binding to the DNA is based on a 34 amino acid repeat motif.

14. TYPICAL STRUCTURE OF TALs Central repeat domain Number of repeats 1.5 to 33.5 Each repeat consists of 34 residues Short repeat at the carboxyl terminal end Hypervariable residues : position 12 and 13. Structure: two alpha helices and a short RVD Typical repeat sequence: LTPEQVVAIASNGGGKQALETVQRVLCQAHG

15. HOW DO TALENs WORK : MECHANISM

16. CRISPR VS TALEN

17. Thank you