New gene-editing instruments delete lengthy stretches of DNA | Spectrum
Restoring function: unlike a traditional CRISPR system (left), a new method (right) can delete a section of DNA and replace a missing part of a gene, restoring expression of the gene in the liver of mice (arrows) .
Two new methods make it possible to delete long sections of the genome and thus expand the capabilities of the gene editor CRISPR. The techniques could lead to therapies that remove large insertions or duplications associated with autism, such as the DNA repeats that underlie Fragile X Syndrome.
To remove a segment of DNA, CRISPR systems typically use an enzyme called Cas9 to cut double-stranded DNA at two target sites. The cell’s own repair machinery can then join the cut ends and omit the sequence in between. However, this process is error-prone and can insert or delete unintended sections of DNA, so-called “indels”, or rearrange large sections of the genome. Cutting off double-stranded DNA can also lead to cell death.
Another CRISPR-based system called “Prime Editing” can make DNA repair more precise. In one version of the technique, a protein complex called the Prime Editor cuts only one strand of DNA at one point and the opposite strand at the other. The Prime Editor will add a sequence to one of the cut strands to guide the repair.
Similar to traditional CRISPR systems, prime editing uses a strand of RNA to guide the editing machinery to a target sequence. But this segment of RNA – called pegRNA – also provides a template for DNA extension, which typically binds to a complementary sequence on the opposite strand. With longer deletions, the DNA extension may be too far from its target to direct repair, and so the researchers were only able to delete sequences less than 100 bases or “letters” in length.
The two new techniques allow researchers to erase up to 10,000 letters of DNA. Both were described in Nature Biotechnology in October.
Paired prime number:
In the first method, the researchers used a pair of pegRNAs to guide the processing machinery to cut target sites on opposite strands of DNA and extend each strand with a specific sequence. They argued that a second DNA extension could help guide the first to its binding site in the genome. The team programmed the extensions to be complementary to DNA on either side of a sequence they were trying to delete. When the appropriate strands joined, the sequence in between was deleted.
To test the system, the researchers created pairs of pegRNAs that were supposed to delete different sequences – between 24 and 10,204 letters in length – and delivered them to a batch of cultured human cells along with a first-class editor. For comparison, they also used a conventional Cas9 enzyme to perform the same deletions in a separate batch of cells.
The new method, called PRIME-Del, worked just as efficiently as the traditional CRISPR system and resulted in significantly fewer errors, the researchers reported. When they deleted a 10,204 letter sequence with PRIME-Del, only 3 percent of the sequences near the edited site contained indels, compared to more than half of the sequences edited through Cas9.
The researchers were also able to insert stretches of DNA at the junction of the deletion. In one test, they designed five pairs of pegRNAs that were programmed to delete part of a gene in a DNA loop called a plasmid. Each pair also encoded a sequence 3 to 30 letters in length that should be inserted into the plasmid at the edited location.
Deleting and inserting sequences at the same time caused minimal errors and was just as efficient as deleting DNA alone, the team reported. Using this approach, researchers could introduce molecular tags into the genome or avoid accidentally translating a gene into protein when DNA is deleted.
PRIME-Del is also less damaging to cells than traditional CRISPR systems because it does not cause double-stranded DNA breaks. Scientists could combine the approach with assays that introduce a variety of mutations into cells to study their effects, the researchers say. The team wants to investigate how regulatory regions of the genome contribute to autism in this way.
Cut and Paste:
Another research team developed a different way to delete and insert DNA sequences. Their approach, called PEDAR, replaces one sequence in the genome with another.
As with the first approach, the researchers used a pair of pegRNAs to escort the processing machinery to two sites flanking a segment of DNA that they wanted to delete. However, a modified Prime editor cut both strands of DNA instead of one. The editor then added complementary single-stranded DNA encoding a new sequence to opposite strands that directed repair and replaced the flanked sequence.
The team constructed a pair of pegRNAs to delete a 991-letter portion of a gene and replace it with an 18-letter sequence. They then combined the pegRNAs with either the modified Prime Editor, a conventional Prime Editor, or a Cas9 enzyme and assessed the performance of each system.
Only the modified Prime Editor deleted and inserted the target sequences, although the protein complex produced unintended changes at a rate comparable to Cas9. Further tests showed that PEDAR can also process longer sequences, insert segments with a length of up to 60 letters and delete around 10,000-letter sequences.
In another experiment, the team used PEDAR to edit a gene in a mouse model for tyrosinemia, a disease caused by mutations in a gene that codes for an enzyme that can lead to liver failure. In the mouse model, a sequence longer than 1,000 letters replaces a 19-letter segment of the gene and disrupts its function.
Using PEDAR to delete the long insertion and replace the missing segment, the researchers restored the gene’s function. Antibody staining of mouse liver tissue a week after treatment showed cells expressing the missing enzyme, the researchers reported.
The new method could be used to develop gene therapies for a wide variety of disorders, say the researchers. They are working to optimize the efficiency and accuracy of the system.
Quote this article: https://doi.org/10.53053/DJPR5209