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Researching CRISPR

In early 2013 a new tool was ingeniously adapted to an immune strategy using bacteria and archaea to protect itself against viruses, judged on the potential to revolutionize the way manipulated mice were made. The name of the new technology was CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and most mouse genetics had never heard of it.

Before 2013, the engineering of mice was a laborious process of multiple steps consisting of genetically altering embryonic stem cells of mice (ES), injecting them into an embryo and breeding several generations of animals. Even the best researchers could take two years to make a mouse with this technology. CRISPR replaces all this with a molecular complex that can perform targeted genetic surgery on a fertilized egg cell and produce a strain of transformed mice over a period of 6 months.

Most researchers receive their manipulated mice from colleagues or by buying commercial outfits or academic repositories. Tailoring a genetically engineered mouse through the industry can run anywhere from $ 40,000 to $ 60,000 or more using the older technology. By making the engineering of mice much easier and cheaper, CRISPR clears the way for more laboratories to make custom models themselves. When you knocked out mice, you needed some skills, says Rudolf Jaenisch of the Massachusetts Institute of Technology (MIT) in Cambridge. Now you do not need them anymore. Any idiot can do it.

But can an idiot do it?
Some mouse engineers have once again pondered the haste to completely replace ES cell technologies with CRISPR. Few doubt their potential, but technology is still a work in progress and the ability to change has a big difference. Although CRISPR easily disables genes, it is less efficient at inserting or breaking new DNA. This is important, not only to give an animal a new function, but also to create a knock-in, known as a conditional knock-out, an animal model in which researchers use a target gene for life at specific times. or disable specific tissues. .

Because CRISPR is less good at making conditional knockouts, William Skarnes, who led a team that makes murine mutant ES cells at the Wellcome Trust Sanger Institute in Hinxton, feared that the new approach would be too stressful. The decision to leave the ES resource and make simple bangers is a mistake, says Skarnes. You still want to create conditions via the ES route.

CRISPR researchers are now refining the technique to do knock-ins more efficiently. But that means tinkering with the mechanisms that cells use to repair broken DNA, which is essential for their health. Some are cautious about overcrowding of biology to increase efficiency.

MIT's Jaenisch was the first to show the strength of CRISPR for producing knockouts from mice. He and his colleagues reported that the technique successfully disrupted five genes in a single set of ES cells from mice, something that was not possible before. More importantly, they showed that they could bypass ES cells altogether and at the same time disable two genes in single-cell mouse zygotes or fertilized eggs. No longer do researchers have to modify ES cells and accurately breed several generations of mice to produce an animal carrying the mutant gene in its egg or sperm cells. And researchers who wanted mice with two mutations would no longer have to cross and continue as mutantsan a time-consuming, cumbersome process.o come to the offspring with the altered germ line. Since then, more than 500 articles have worked out how CRISPR can disable mice and disable genes.

The impact of CRISPR is measured in more than in savings. The ease and speed of the technology makes it possible to engineer mice on the fly, to solve new and specific target puzzles and also to adapt genetically modified mice in other, new ways.

On the one hand, however, the CRISPR revolution faltered. Three months after the first CRISPR report from his laboratory, Jaenisch and his colleagues published a second article in Cell that suggested that CRISPR could easily perform more complex genetic surgery by tapping pieces of DNA instead of simply turning off genes. As a demonstration, they used CRISPR to knock fluorescent tags into mouse zygotes, which emphasized when a specific gene was switched on. They also created conditional mutants, which are crucial for many research efforts. Using CRISPR to insert loxPs into zygotes, the Jaenian team reported that it made conditional mice with relatively high efficiency - about 16% of zygotes led to mouse puppies with the right mutations.

Gene editing service https://www.tebu-bio.com/blog/2018/05/21/gene-editing-tunr-system-to-remix-the-gene-expression-level/

Skarnes is one of the many researchers who are overwhelmed by the first reports of Jaenisch, but he was disappointed when he tried to use the technique in his own laboratory. It was clear from his papers that this would be easy and I was pretty sure that this would make ES unnecessary, says Skarnes. What was disappointing was that none of us could reproduce with the efficiencies reported by Jaenisch. ... It works at 1% or 2% and many projects fail. It is not really proven that it is a robust method.

Skarnes calls the transition to CRISPR for conditional knockouts. But he admits that researchers will eventually discover how CRISPR can be adapted to make conditional mutant mice with high efficiency. Regardless of the shortcomings of CRISPR at this time, the potential for engineering mice should not be underestimated. There are things that CRISPR can do that people are just starting to understand. It is in the first stages of development and the tool has endless possibilities.