Super-muscly pigs created

by small genetic tweak

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Masume mohamadian

Jin-Soo Kim, a molecular biologist at Seoul National University

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MSTN

Cytogenetic Location: 2q32.2

The official name of this gene is “myostatin.”

• Transcription activator-like effectorsTo introduce this mutation in pigs, Kim used a gene-editing technology called a TALEN, which consists of a DNA-cutting enzyme attached to a DNA-binding protein. The protein guides the cutting enzyme to a specific gene inside cells, in this case in MSTN, which it then cuts. The cell’s natural repair system stitches the DNA back together, but some base pairs are often deleted or added in the process, rendering the gene dysfunctional.

TALEN

Structure of TALEN

• In nature, TALs are DNA binding proteins from bacteria that infect plants • DNA binding is mediated by 34 bp

amino acid repeats, which differ at amino acids 12 and 13. These “repeat variable diresidues”, or RVDs • Each RVD binds one nucleotide

Nuclease domains are FOK 1 s

Repair

The first is homologous recombination (HR) The second major mechanism for DSB repair is

nonhomologous end joining (NHEJ) double-strand breaks (DSBs)

CRISPR/Cas9the team is now doing the same experiment with another, newer gene-editing technology called CRISPR/Cas9

CRISPR/ Cas system

CRISPR/ Cas modules are adaptive immunity systemsthat are encoded by most archaea and many bacteria andthat act against invading genetic elements, such as phageand plasmids40%bacteria and 90% archaea

• CRISPR= Clustered Regularly Interspaced ShortPalindromic Repeats

• Cas= CRISPR-associated proteins

CRISPR/ Cas systemThere are 3 steps in the CRISPR–Cas invaderdefense pathway:

• 1. Adaptation• 2. RNA processing (CRISPR RNA biogenesis)• 3. RNA interference (Invader silencing)

Which should I use, TALEN or CRISPR?

recent improvements in the technology, such as the use of double nickases, truncated guide RNAs, and a Cas9-Fok I fusion, improve CRISPR target site specificity.

RNAi and genome editing• RNAi-mediated knockdown is the most common

strategy for ablating gene function in higher eukaryotes. However, there are a few key differences between RNAi and genome editing. First, RNAi does not completely shut off the gene . Rather, gene expression is down-regulated post-transcriptionally, without changing the genetic code. Some functional RNA or protein remains and is translated. So, the RNAi strategy is a “knockdown”. Gene function is reduced, not eliminated. In genome editing, on the other hand, the genetic code is changed, and attenuation of gene expression is usually complete, leading to a “knockout”. The decision on whether to use RNAi or genome editing for attenuating gene expression depends on the goals of the experiment.

References

• Super-muscly pigs created by small genetic tweak. Jin soo kim at nature

• ZFN, TALEN and CRISPR/Cas-based methods for genome engineering .Thomas Gaj

• Genome Editing Technology From GeneCopoeia, Inc. Ed Davis

• Editing our DNA with Molecular Scissors at The Tech Museum of Innovation

• RNA-Guided Endonuclease at toolgene.com

• Advances in genome editing technology and its promising application in evolutionary and ecological studies. Lei Chen