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Genome Editing

Platinum TALEN

TALEN (Transcription Activator-Like Effector Nuclease) is a transcription factor-like effector protein (TALE) from plant pathogenic bacteria fused with the nuclease domain of FokI, a type of restriction enzyme.

The DNA-binding domain, TALE, contains a DNA-binding repeat consisting of a series of 34 amino acid modules, each of which can recognize a single base of DNA. The 12th and 13th amino acids in each module are called RVDs (Repeat Variable Diresidues), and by changing the amino acid composition of the RVDs, the DNA bases that can be recognized are changed. Using this property, TALEs that specifically bind to target DNA sequences can be created by assembling DNA-binding repeats with appropriate RVDs.

Unique Genome Editing Technology

CRISPR-cas9 has become widely used in the world due to its usefulness and ease of production. However, the risks associated with expensive licensing fees, especially in the medical field, and the high cost of communication with regulatory authorities related to the Cartagena Act because of the use of nucleic acids have become challenges for industrial applications.
PtBio is promoting social implementation of ZFN and TALEN, which are suitable for industrial applications because the licenses are organized and can be applied without nucleic acids.

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Superior DNA binding (specificity) due to cyclic repeats

Conventional TALENs generally use fixed amino acid sequences other than the RVD in each module, but Platinum TALENs have realized higher DNA binding properties than conventional TALENs by focusing on the fact that there is diversity even in the 4th and 32nd amino acids in the module in natural TALE proteins and by introducing periodic diversity in these positions. By introducing cyclic diversity in the amino acids at these positions, the system achieves higher DNA binding properties than conventional TALENs*1.

1 So far, the system has been confirmed to have extremely high cleavage activity in cultured cells, including human iPS cells, and in all kinds of cells and organisms, including nematodes, Daphnia magna, sea urchins, ascidians, zebrafish, newts, frogs, mice, rats, and marmosets*2,3.

Simplified and Efficient Production by Platinum Gate System

It is generally believed that TALEN is a more difficult tool to manufacture than CRISPR and not everyone can use it.
PtBio has established its own system, the Platinum Gate system, which enables efficient construction of Platinum TALENs*1.
Furthermore, we have established a library of Platinum TALENs that can be used for all types of target sequences, and are ready to efficiently provide Platinum TALENs for industrial applications.

*1 Sakuma T et al., Sci Rep, 3, 3379 (2013). 
*2 Sakuma T & Woltjen K, Dev Growth Differ, 56, 2-13 (2014).
*3 Sakuma T & Yamamoto T, Methods Mol Biol, 1630, 25-36 (2017).

FirmCut Nuclease Domain(ND1)

Nuclease domain derived from Firmicutes, identified as a novel DNA cleavage domain derived from a species different from conventional FokI.

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Excellent cleavage activity and convenience in purification

It has various advantages over conventional FokI, such as high cleavage activity to the target sequence and dramatically improved yield of recombinant protein.

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Reduction of off-target risk

When cleaving a target sequence, the restriction enzyme forms a dimer (heterodimerization) and activates only the specific dimer combination, thereby enabling "two-step" sequence cleavage. This reduces off-target risk.
Unlike conventional Fok I, ND1 has been shown not to decrease cleavage activity even in heterodimeric mutants with increased specificity for the target sequence, and is expected to be applied in the medical and other industrial fields.

 

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Gene transfer system PITCh system

Genome editing is performed using DNA repair mechanisms, and there are three main repair mechanisms: HR, NHEJ, and MMEJ.
In general, homologous recombination (HR) repair mechanisms are used to introduce (knock-in) target genes into targeted regions on chromosomes, but this is difficult to do in organisms with poor HR ability.

 

Gene Editing of Difficult-to-Transfer Species Now Possible

PtBio's CTO, Takashi Yamamoto, and Science and Technology Advisor, Satoshi Sakuma, and their colleagues have established a simple method to create TALEN vectors for gene transfer by shortening the base sequence to be bound to both ends of the transgene*1,2,3.
There have been many examples of TALEN vectors being applied to organisms that have been difficult to transduce, and it has been shown that TALEN vectors can be used for gene disruption and gene transfection at the same time.

 

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Transgenesis or Knock-in/knock-out

*1. Nakade S et al., Nat. Commun. 5, 5560 (2014).
*2. Sakuma T et al., Nat. Protoc., 11, 118-133 (2016). 
*3. Kazuki N et al., Bioeng. 8(3), 302-308-(2017). 

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