Human embryonic kidney cells turn green after repairing a damaged DNA induced by CRISPR with a PCR fragment encoding a fluorescent protein and homology with 33 nucleotides. .
Credit: Alexandre Paix
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Johns Hopkins scientists have developed a streamlined approach and combined with “standard” efficiency for the introduction of new DNA sequences in cells after using the gene-cutting tool known as CRISPR. . The scientists say the method, which they based on experiments with mouse embryos and thousands of human cells, could improve the consistency and efficiency of genome editing.
The new method and its progress are described online on November 28th Methods of the National Academy of Science.
“CRISPR is a tool to help scientists modify the genome, predict the outcome of certain traits and study them, but the tool itself only creates genome destruction. It can’t control how it’s inserted in a new DNA sequence in the genome, ”he added Geraldine Seydoux, Ph.D., the Huntington Sheldon Professor of Medical Discovery in the Department of Molecular Biology and Genetics and vice dean for fundamental research at Johns Hopkins University School of Medicine, and an investigator at Howard Hughes Medical Institute.
“We set out to study how CRISPR-induced cell repair with the aim of using the cell’s natural DNA repair process to introduce new genome sequences. We were surprised to find that cells can easily copy sequences from foreign DNA to repair DNA damage, as long as the foreign DNA is linear, ”Seydoux added.“ By studying how to copy. the foreign fragments of DNA during the repair process, we got some simple rules to make as efficient editing of the genome as possible, optimize the use, and do it with confidence. “
CRISPR, which stands for clustered regularly interspaced short palindromic repeat, has gained popularity among scientists over the past five years as a tool to efficiently break down DNA. It is adapted for use in mammalian cells from a natural process of viral protection in bacterial cells involved in the creation of lethal cuts in viral DNA. Basically, the tool is a streamlined set of “scissors.”
The continuing belief, among scientists, is that cells repair DNA breaks by inserting an internal set of nucleotides, the chemical blocks of DNA. It can usually destroy any gene that is in the area where the DNA is broken.
Scientists also know that, sometimes, cells use a different source – a sequence from another piece of DNA, or “donating” DNA – to seal DNA damage. However, the new “donating” order cannot insert itself into a vacuum.
However, the newly donated DNA requires a type of tape at each end to help it retain the texture created by the edge. Scientists refer to this tape as the “homology” arms of the donor DNA.
The homology arms consist of nucleotides that overlap the inner parts of DNA with the same genetic code. This helps the donor “stick” to the will of the DNA.
However, scientists regard the use of donor DNA as an inefficient way to repair the genome, considering that it requires long arms of homology, especially if a long sequence of DNA, and either hard or round DNA, which is difficult to prepare at large sizes.
While the scientists have gained a lot of experience with CRISPR, Seydoux said, “Questions have arisen about the best design rules for donating DNA and arm length homology.”
Seeking answers to these questions, Johns Hopkins scientists have incorporated different combinations of donor DNA into human embryonic kidney cells, known for their ability to grow well and of frequent use in cancer research. The scientists used donor DNA with a gene that writes for a fluorescent protein, which glows green on the cell’s nuclear membrane if the gene is successfully inserted.
Johns Hopkins research fellow Alexandre Paix found that DNA fragments work very well as well as donors, and are two to five times more efficient than round DNA (known as plasmids). ) in human cells. “Linear DNA is very easy to prepare in the laboratory, using PCR,” says Paix, who refers to polymerase chain reaction devices, which are used to amplify DNA.
Paix also tested different arm lengths for homology. He found that the sweet spot for the arms of homology was about 35 nucleotides in length, much shorter than is commonly used by scientists.
Specifically, it has been found that homology arms of 33 to 38 nucleotides are just as successful as those with 518 nucleotides, providing between 10 and 20 percent successful edits under the most very good condition. In contrast, when scientists tested homology weapons that were 15 and 16 nucleotides in length, the rate of penetration success fell by half. They repeat these results at three different locations in the human genome.
They also know that the newly inserted sequence, with innumerable homology arms, can be up to 1,000 nucleotides in length.
The team obtained success rates between 10 and 50 percent with coverage ranging from 57 to 993 nucleotides in length. Shorter rows are more successfully inserted than longer ones. For example, the new sequences 57, 714 and 993 nucleotides were successfully inserted 45.4, 23.5 and 17.9 percent of the time, respectively. With more than 1,000 nucleotides, the new intrusions with 1,122 and 2,229 nucleotides were less successful-about 0.5 percent of the time. “At that size, it’s difficult to identify the amount of donor DNA needed for editing. Cells have a tendency to be ‘stung’ by a lot of DNA,” Seydoux said.
Finally, the team also found that the success rate of editing peaks when the new sequence was positioned within 30 nucleotides from the truncated CRISPR site. “With more than 30 nucleotides, the penetration doesn’t work,” Seydoux said.
“These parameters should accommodate most of the genes that scientists wish to edit. In fact, most experiments involve editing only two to three nucleotides close to the CRISPR cut area,” he said. added Seydoux.
The research team also tested whether the same method could function in mouse embryos. Using a PCR fragment with 36-nucleotide homology arms, the team successfully filmed a 739 nucleotide high-sequence coding for a fluorescent protein in 27 of 87 (31 percent). ) mouse embryos.
Seydoux’s research team is already using healing rules to study DNA Caenorhabditis elegans, a class of wate, and the researchers studied whether the healing rules were included in other types of human cells.
Before the guidelines were adopted, Seydoux said they had to be tested on many cell types in humans and other organisms.
Other scientists who contributed to this research included Andrew Folkmann, Daniel Goldman, Heather Kulaga, Michael Grzelak, Dominique Rasoloson, Supriya Paidemarry, Rachel Green and Randall Reed from Johns Hopkins University.
The research was funded by the Howard Hughes Medical Institute and National Institutes of Health (provided R01HD37047, R01DC004553 and F32GM117814).