CRISPR-Cas3: error-free editing
Scientists have figured out how to make a new genomic editor safe for people
Biologists for the first time received atomic "photos" of one of the versions of the CRISPR/Cas genomic editor and discovered an error correction system in it, modification of which will make it safer for humans, according to an article published in the journal Cell (Xiao et al., Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System).
"In order for us to use CRISPR in medicine, we must be completely sure that this system works with 100% accuracy and that it does not edit the wrong genes that it should change. We found that the CRISPR-Cas3 system has such a trait, and now we know how to change its operation if errors still occur when editing the genome," explains Ailong Ke from Cornell University (USA).
Molecular Typos
The CRISPR/Cas9 genomic editor, called the major scientific breakthrough of 2015, was discovered by American scientist Feng Zhang and a number of other molecular biologists about four years ago, and since then it has undergone several upgrades that allow scientists to use it to edit the genome with ultra-high accuracy.
In fact, CRISPR/Cas9, like many other things, was invented not by man, but by nature – initially this system developed inside bacteria hundreds of millions of years ago to protect against retroviruses. It consists of two components – a "library" of samples of the genetic code of viruses (CRISPR), and the Cas9 enzyme, which searches for similar sequences in the DNA of a bacterium and removes them if necessary.
In 2013, the rapid development of this technology began, and today it has been used to edit the genomes of dozens of living beings, including human embryos, as Chinese geneticists confessed in April 2015. These experiments revealed the main drawback of CRISPR/Cas9 – the editor, especially with multiple gene changes, sometimes made mistakes and deleted unnecessary DNA segments.
Such behavior is permissible during genetic experiments in the laboratory, but is unacceptable in medical practice, where such a typo can cost the life of a person in whose DNA a "surgical intervention" is performed.
Genetic Seamstress
Ke and his colleagues found a potentially quick way to add a system for correcting such "typos" to CRISPR/Cas9 by studying another similar bacterial "antivirus" – the CRISPR/Cas3 system. Its main difference from CRISPR/Cas9 is that it does not just remove viral DNA from the bacterial genome, but destroys those DNA sequences whose "templates" are in the antiviral genetic "database" of the microbe.
In the past, this system did not attract the attention of scientists because it could not be used to remove small segments from the DNA of patients and replace them with correct sequences of genetic "letters"-nucleotides. It turned out that it had built-in systems for "verification" of deleted sequences that protect the cell from CRISPR/Cas3 destroying the microbe genome itself by mistake.
To detect these systems, scientists first had to cool the CRISPR/Cas3 molecules to temperatures close to absolute zero, and then use a special electron microscope to take photos of them at an almost atomic level.
A sample image of CRISPR molecules using a cryo-electron microscope (left). The research team combined hundreds of thousands of two-dimensional images (right) to obtain a three-dimensional scheme of the gene editor. In the figure below, an RNA molecule is highlighted in blue, orange and red are partially unwound DNA strands. Drawings from the Harvard Medical School press release Bringing CRISPR into Focus - VM.
These images showed that the correction system in CRISPR/Cas3 works in a fairly simple way. First, it looks for a sequence in the analyzed DNA similar to the one contained in one of the templates from the CRISPR "library". If she finds a similar set of "letters"-nucleotides, then she bends one of the two strands of DNA, "tearing" it from the second strand, and tries to connect it to the template.
If this can be done, the CRISPR/Cas3 system changes its structure, pulling the second strand of DNA in a similar way, and allows the Cas3 protein to begin their destruction. If the template and the DNA strand do not completely match, then the loops will be asymmetric, which will not allow Cas3 to destroy them. This protects the cell from accidentally destroying other parts of the DNA, for some reason similar to how the virus genome works.
Studying how this part of CRISPR/Cas3 works, scientists hope, will help their colleagues understand how to make CRISPR/Cas9 safer for editing the human genome for therapeutic purposes.
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30.06.2017