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TALEs......... Transcription activator like effectors These are proteins that bind to DNA in a sequence-specific way Secreted by bacterial pathogens into plant cells These enter the nucleus and activate expression of individual genes Pathogens like Xanthomonas and Ralstonia known to secrete TALEs Plays a crucial role in determining the bacterium to infect the host 3


TALENs....................... These are novel classes of sequence specific nucleases Created by fusion of TALE domain to the catalytic head of endonulcease functional domain Used to create site specific gene modification in plants , yeasts, animals and even in human cells ZFNs and meganucleases are also used for targeted genome editing 4

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Tale of TALENs:

Tale of TALENs AvrBs3 is first TALE identified from X.compestris Ulla Bonas et.al., 1992 DNA recognition specificity of TALEs Boch and Bogdanove . 2009 First TALEN developed Bogdanove . 2009 Xanthomonas consist 28 TAL genes per strain 6

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TALEN acts as a heterodimer (two units of a TALE DNA binding domain fused to a catalytic domain), cleaving two close sequences, and thereby increasing specificity to a complexity of 30-40 base pairs. 7

Components of TALENs:

Components of TALENs 8

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TALE domain:

TALE domain N-terminal domain : Type three secretion signals Nucleotide localization signal : Diverts the TALE inside the nucleus Acidic activation domain : Activates the plant gene expression Central repeat domain : DNA binding specificity 11

Central repeat domain:

Central repeat domain It is highly conserved region Consists of varying number of repeat units ranges from 1.5 to 33.5 in different pathovars of Xanthomonas spp. Each tale consists of 33 to 35 amino acids repeats that confers specific DNA sequence recognition to the TALE protein Repeat variable Di-residues (RVD) : repeat units with high level of identity with the exception of two variable amino acids at position 12 and 13 12

Repeat variable Di-residues:

Repeat variable Di-residues It confers DNA binding specificity to the repeat region At least two dozens unique RVDs are found There are seven sequences which are common – HD, NG, HG, NN, NS, NI, and N* N* - 33 residue repeat with a missing residue within RVD loop 13

Catalytic domain of an endonuclease :

Catalytic domain of an endonuclease It induces the DNA double strand breaks (DSBs) in vitro and in vivo These breaks are repaired by two peculiar mechanisms Non homologus end joining (NHEJ) Homologus reombination (HR) Generation of DSBs at specific loci can be utilized to perform a variety of modification including gene deletion, additions, corrections 14

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ARTIFICIAL TALENS....( dTALENs ) Based on the specificity of RVDs in each repeats, TALENs were designed artificially by Golden gate cloning method Companies which produces Artificial TALENs are Cellectis bioresearch Life technologies FLASH assembly Modular assembly 17

Applications of TALENs for targeted genome editing in various organisms.:

Applications of TALENs for targeted genome editing in various organisms. 18

Applications of TALENs:

Applications of TALENs 19

Advantages of TALENs........:

Advantages of TALENs........ 20



Future thrust areas:

Future thrust areas 22

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