A hidden component of the CRISPR/Cas3 genome editing system, which is essential for it to work, has been discovered by scientists.
The CRISPR/Cas3 family of genome editing systems is similar to other CRISPR families but can uniquely make larger and more accurate changes to DNA (see BioNews 1069). While researching a new variant of the more efficient CRISPR/Cas3 system, researchers at the University of Michigan identified an additional protein called Cas11 that is necessary for CRISPR/Cas3 to make edits.
'This type of Cas11 protein was like a hidden gem that's been only recently noticed by us and other researchers' said Dr Yan Zhang, the lead author of the study.
Like other CRISPR systems, which are derived from bacterial immune mechanisms, CRISPR/Cas3 forms complexes based on protein and RNA that are able to cut DNA. Unlike the more commonly used CRISPR/Cas9 systems where only a single Cas9 protein subunit is needed, CRISPR/Cas3 complexes require a variety of Cas proteins, including Cas3, to co-assemble.
In the study published in Molecular Cell, scientists purified CRISPR/Cas3 complexes directly from the bacteria Neisseria lactamica. These complexes were able to edit the DNA in human cells and stem cells grown in a laboratory with 95 percent and 50 percent efficiency respectively. According to Dr Zhang, using the older CRISPR/Cas13 system 'we were only achieving 10 percent editing in stem cells and maybe 30 to 50 percent editing in cancer-derived cell lines.'
Researchers tried to recreate this system by inserting the genes for the Neisseria lactamica CRISPR/Cas3 components into cells in the form of small circular DNA strands called plasmids. However, no genome editing was observed. After going back and studying the protein components of the CRISPR/Cas3 complexes, an additional, previously unreported, protein was discovered that was named Cas11.
The gene for Cas11 was found located within the gene for another CRISPR/Cas3 component, Cas8. An alternative translation initiation site was enabling production of the Cas11 protein from bacterial RNA instead of Cas8 – a process called internal translation. When an additional plasmid containing the Cas11 gene was applied to human cells alongside the other CRISPR/Cas3 components, genome editing was reactivated.
CRISPR systems can be programmed to repair DNA with a specified template after cutting DNA. Compared to CRISPR/Cas9, CRISPR/Cas3 facilitates these repairs with a greater specificity, which has been a concern surrounding potential use in patients for gene therapy. Furthermore, the larger size of deletions CRISPR/Cas13 performs opens the possibility of replacing entire genes for therapy or biomedical research.
'I think we found the version of CRISPR/Cas3 tool that we are very satisfied with' said Dr Zhang. 'The next phase for us is to apply CRISPR/Cas3 in animal models and stem cells to create disease models'.
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