Question

comparison between ZFNs , TALENs and CRISPR/Cas9 Genom Editing Systems

Answer 1

Ans . ZFN (Zinc finger nuclease) :- These domains are relatively small protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecules.

The first endonucleases were ZFNs( zinc finger nucleases). These are based on zinc finger proteins, a family of naturally occuring transcription factors, fused on an endonuclease Fokl1.

Zinc finger domains can recognize a trinucleotide DNA sequence. A series of linked zinc finger domains can , therefore , recognize longer DNA sequences, providing desired on-target specificity.

However , zinc finger motif aligned in an array influence the specificity of neighboring zinc fingers, making the design and selection of modified zinc finger arrays more challenging and more time consuming. It is hard to predict the specificity of final arrangement.

TALEN (Transcription activator-like effector nuclease) :- These are fusion proteins of a bacterial TALE protein and Fokl endonuclease. Similarly to ZFNs , target specificity comes from protein-DNA association. In the case of TALENs , a single TALE motif recognizes one nucleotide and an array of TALEs can associate with a longer sequence.

The activity of each TALE domain is restricted only to one nucleotide and does not affect the binding specificity of neighboring TALEs, making the engineering of TALENs much easier than ZFNs.

CRISPR-Cas9 ( clustered regularly interspaced short palindromic repeats) :- This system is based on bacterial immune system. It is composed of Cas9 nuclease and two types of RNAs : trans-activating crRNA (tracrRNA) and a single guide RNA (sgRNA) that recognizes the target sequenceby standard Watson-Crick base pairing. It has to be followed by a DNA motif called a protospacer adjacent motif (PAM). Each Cas9 protein has a specific PAM sequence.

DNA cleavage is performed by Cas9 nuclease and can leads to a double - strand break in the case of a wild type enzyme, or to a single - strand break when using mutant Cas9 variants called nickases.

This offers many advantages over ZFNs and TALENs , including easy design for any genomic targets, easy prediction regarding off-target sites, and the possibility of modifying several genomic sites simultaneously.