TALENs (Transcription Activator-like Effector Nucleases)

TALNs are Restriction enzymes which use as Genome editing tool,

1. Transcription Activator-like Effector Nucleases. (TALEN’s) Muhammad Mujahid ID: F2019253023

2. Transcription activator-like effector nucleases ( TALEN ) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing of: DNA-binding domain (TAL effector ). DNA cleavage domain (a nuclease which cuts DNA strands ). INTRODUCTION.

3. TALEs TALEs – Transcription Activator-Like Effector proteins produced by certain plant pathogenic bacteria, Xanthomonase spp. Act as effectors during pathogenesis . It contain: NLS (nuclear localization sequence). N-terminal translocation signal. Transcriptional activator domain. Central repeat domain. Contain 17 to 18 tandem repeats of a 33-35 amino acid motif that can recognise a single base through two di-variant residues DNA binding specificity is determined by the amino acids at positions 12 and 13 within each repeat which is called R epeat Variable Diresidue ( RVD) .

4. TALEs .

5. 5 Structure and DNA binding code of TALEs NG = T HD = C NI = A NN = G or A DNA binding code N’ C’ Central repeat domain AD NLS L T P E Q V V A I A S H D G G K Q A L E T V Q R L L P V L C Q A H G 1 34 12 13 T S Repeats (1.5 to 33.5) Repeat variable diresidue (RVD) 12 13

6. 6 How to engineer TALEs? N’ T S C’ AD NLS HD HD HD NI NI NI NG NG NG NK NI NG HD NK DNA Target T CCC AAA TTT G A T C G Engineered central repeat domain = C HD NI NG NK = A = T = G

7. TALENs

8. G G A T G N N N N N N N N N N N N N N N N N N N C C T A C N N N N N N N N N N N N N N N N N N N 5’ 3’ 3’ 5’ Nuclease DNA-binding 21-N o v -16 8 Fok I Flavobacterium okeanokoites N-terminal: DNA-binding domain C-terminal: nonspecific DNA-cleavage activity

9. Fok I nucleases 21-N o v -16 9

10. Cleavage of DNA

11. Genome editing.

12. Transcription activator like Effector Nucleases (TALENs) is a synthetic modified Restriction Enzyme. It consist on TALEs (proteins) and Nuclease enzyme (Fok 1). It use as tool for Genome Editing. In 2011, named “The methods of the Year” Conclusion.