TALEN technology

(Daniela Penna)

GeneArt TAL or TALEN technology is derived from TAL proteins.

Transcription Activator–Like (TAL) effector proteins are produced by Xanthomonas bacteria, a genus of plant pathogens.

TAL naturally binds to specific sequences of the host DNA, altering the gene expression. TAL proteins have two distinct domains: an effector domain and an extraordinarily specific DNA-binding domain.

The DNA-binding domain consists of a variable number of amino acid repeats. Each repeat contains 33–35 amino acids. The amino acids number 12 and 13, called Repeat-Variable Di-residues (RVDs), are responsible for the specific recognition of a single DNA base pair.

The DNA-binding domain repeats can be assembled modularly, and the RVDs can be varied to create a TAL protein that specifically targets any locus in the genome.

The DNA-binding domain can then be linked to a custom Effector domain (e.g., a nuclease, or a transcription activator or repressor) to create a chimeric protein to precisely manipulate DNA.

This technology is known to function in bacteria, yeast, plants, insects, zebrafish, and mammals.

The TAL effector can:                                   

• Knock out a gene
• Correct a gene
• Insert a DNA sequence
• Activate a gene transcription
• Repress a gene transcription
• Any other function of your choice

 TALEN technology can be used to:

• Elucidating gene function and regulation
• Metabolic pathways study
• Embryonic stem cell research
• Disease models research

To better understand, watch this video made by Thermo Fisher: 

How TALEN works

CRISPR-Cas9 technique

(Olocco Federica)

Images taken from http://www.addgene.org/

Crispr/cas9 is a new technique that was developed studying the adaptive immune system of bacteria and archea against phages and other foreign genetic elements.
In particular, the most interesting proteins are clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (Cas), constitute of the adaptive immune system.

The system in bacteria and archea is complex  and different steps are involved:

• a CRISPR array contains many unique protospacer sequences, each protospacer is an homologous to foreign DNA, that are separated by short palindromic repeat sequences.
• The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA), a long transcript cantaining all the protospacer sequence separated by the palindromic repeat
• The pre-crRNA is processed, into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit RNAse III and Cas9 enzymes, which together separate the individual crRNAs. In this way, each crRNA contains only one unique protospacer.
• Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complementary to the crRNA
• After the complex binds, Cas9 separates the double stranded DNA target and cleaves both strands.

To use this system as tool for genome editing, it has been simplified: a single guide RNA (sgRNA) was developed an it combines the traRNA and the crRNA. So, sgRNA is able to specifically target Cas9. Once the complex seeks put the complementary DNA sequence, Cas9 can separate the double strands and apply the cleavage.

After the cleavage, NHEJ (non-homologous end joining) or HDR (homologous direct repair) pathways occurs in order to joint again the double strand and in this passage,  it is possible to make precise modifications. It is possible to change the reading frame, introduce insertion, deletion.

When compared to the previous technique, such as ZNF and TALEN, CRISPR/Cas9 system is much simpler and flexible. Moreover, it highly increases the efficiency of the genome editing event.

This is only a little introduction to this complex system. Here you can find two video that explain very well how this tool eas discovered and how it works. The first is a Ted Talk from Jennifer Doudna that is a co-inventer of this technique. The title of the video is “How CRISPR lets us edit our DNA”

This second video is made by nature video

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