These two cute little monkeys keep resurfacing wherever I surf, begging for a post, so here it is!
While most of the research on gene therapy involves mice and rats, scientists in China have now succeeded in engineering the worlds first ever genetically modified primates. Rodents have dominated animal model research because of their feasibility – they reproduce much more quickly and in large numbers enabling scientists to gather and analyse data far more quickly.
Nonetheless, primate models are essential in studying human diseases as some disorders in particular are difficult to model in rodents and the results obtained from mice and rats may not necessarily be relevant in humans. Primates are the closest biological species to humans and modelling human diseases in them would probably yield the best insights.
Early attempts to engineer genetically modified primates faced many hurdles. Many of the existing techniques, such as zinc finger nucleases, often resulted in off-target mutagenesis in primates and thus producing genetically modified animals at the desired target sites were impossible. These twin monkeys were engineered with two targeted mutations using the CRISPR/Cas9 system.
The CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) system is a genome editing tool much like the more common zinc finger nucleases. CRISPR is an exciting new player in the field of genome editing that has taken a big leap in the last year. And now, it has become revolutionary in that it has paved the way in preventing off target mutagenesis in primate models.
CRISPR is derived from an a system that bacteria and archaea use as a means to protect themselves against foreign invasive elements.
The system involves a bacterial nuclease, Cas9 which binds to short bacterial RNA sequences called CRISPR RNA (crRNA or guide RNA). In the bacterial system, this RNA is transcribed from host genomic elements previously captured from foreign invaders. These crRNAs then guide Cas9 nucleases to specific locations on a plasmid or the bacterial genome. The crRNA can bind on either strand of DNA and the Cas9 will cleave both strands, resulting in the silencing of that DNA sequence.
When a scientist wants to target a DNA sequence, he needs to insert a short DNA fragment containing that target sequence into a RNA expression plasmid containing a suitable promoter and necessary elements for proper processing in eukaryotic cells. Cas9 nuclease with a nuclear localisation signal (NLS) is expressed from an appropriate promoter and is optimised for expression in various eukaryotic cell types including human. Both the Cas9 expression plasmid and the RNA expression plasmid are transfected into the target cell. The RNA binds and guides the Cas9 nuclease to cleave or replace the target sequence.
This system has been used in gene therapy animal models to either disrupt the gene function or replace it with a new one. The CRISPR approach is far more precise and efficient in comparison to existing gene editing tools. Cas9 is not activated if there is a single base-pair mismatch between the RNA sequence and the genome sequence. Each of the RNA segments can target a different sequence, enabling the nuclease to target one or more positions in the genome. This system limits off target cleavage unlike other gene editing tools as it recognises target sequences of a fixed length and the specificity of Cas9 RNA guided endonuclease.
The scientists from this research tested the CRISPR system in a monkey cell line and were able to disrupt each of the three target genes with 10-25% success. They targeted the three genes in 180 single-celled monkey embryos out of which 83 embryos were implanted. One pregnancy out of 10 that resulted from the implantation led to the birth of the twin monkeys. These monkeys have mutations in two genes: Ppar-γ, which helps to regulate metabolism, and Rag1, which is involved in healthy immune function.
This success is a huge deal for animal model research as it has proven the ability to create a substantial preclinical primate model. Scientists are already working on monkey models for autism and immune dysfunction and this success is most definitely the first step in changing the face of treatments in the future.