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:
i. DNA-binding domain (TAL effector).
ii. DNA cleavage domain (a nuclease which cuts DNA
strands).
3. TALEs
• TALEs – Transcription Activator-Like Effector proteins
produced by certain plant pathogenic bacteria, Xanthomonase
spp.
• Act as effectors during pathogenesis.
• It contain:
i. NLS (nuclear localization sequence).
ii. N-terminal translocation signal.
iii. Transcriptional activator domain.
iv. 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 Repeat
Variable Diresidue (RVD) .
4.
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
ADNLS
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 3412 13
TS
Repeats (1.5 to 33.5)
Repeat variable diresidue (RVD)
12 13
6. 6
How to engineer TALEs?
N’
TS
C’
ADNLS
HD HD HD NI NI NI NG NG NG NK NI NG HD NK
DNA Target TCCCAAATTTGATCG
Engineered central repeat domain
= CHD
NI
NG
NK
= A
= T
= G
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-Nov-16
8
Flavobacterium okeanokoites
N-terminal: DNA-binding domain
C-terminal: nonspecific DNA-cleavage activity
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”
Editor's Notes
Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326(5959), 1501-1501.