While investigating the ancestry of Cas proteins, the researchers also found the first evidence of CRISPR activity in a eukaryotic cell.
CRISPR gene editing is a field with many promises that has sometimes struggled to deliver. Now, a new study published in the journal Science has made a step towards understanding the history of CRISPR’s toolkit, after finding a new editing enzyme among a family of proteins called IscB.
These proteins are thought to be the ancestors of the Cas9, which is often the enzyme of choice in CRISPR.
By using Cas9 alongside a guiding RNA, it is possible to cut a DNA sequence using CRISPR techniques. The use of RNA to guide Cas9 is critical, as it enables versatility in targeting which bits of the genome scientists intend to alter.
The discovery of other RNA-targeted enzymes capable of cutting DNA such as those in this research could yield further tools for genome editing, the study’s authors said.
“These programmable proteins are very useful, beyond basic biological interest,” said lead author Feng Zhang, a molecular biologist at the MIT in Cambridge
“And this mechanism of RNA-guided DNA recognition is likely something that nature has created independently multiple times.”
While researchers have used Cas proteins for genetic engineering, CRISPR is thought to have originated as a microbial defence systems. Found in bacteria and archaea, computational studies suggest that Cas9 likely evolved from proteins in the IscB family. Researchers didn’t previously know what the function of IscB proteins were, however.
‘Every time I give a talk, people always ask me if we’ve seen CRISPR activity in a eukaryotic cell. Now, I can finally say ‘yes’’
–DR FENG ZHANG
Zhang and colleagues found that the DNA responsible for encoding IscB proteins is often located near the DNA that generates a type of RNA molecule called omega RNA. The researchers also found that some IscB proteins can cleave DNA at a site specified by omega RNA in a manner similar to Cas9 and its guide RNA.
A trove of tools
The researchers also investigated another family of proteins called TnpB, which they believe could be the ancestor of the enzyme Cas12, another protein commonly used in CRISPR.
By going through existing databases, the researchers found more than 1m genes that could code for TnpB proteins and provide useful tools for genome editing.
Importantly, the IscB proteins showed up in the database not only in bacteria and in archaea, but also in the chloroplasts inside the cell of an alga. This is the first demonstration of a genome-editing system inside of a eukaryote (the group of organisms whose cells have nuclei).
“Every time I give a talk, people always ask me if we’ve seen CRISPR activity in a eukaryotic cell,” said Zhang. “Now, I can finally say ‘yes’.”
The researchers explained that in nature, these genes could be performing a variety of functions, such as defence or the regulation of other genes’ expression. In the lab, the discovery might translate into a variety of editing tools.
When Zhang and his colleagues tested IscB, they found it cut DNA at a lower efficiency than Cas9. Zhang said that the system has room for improvement, however, and pointed out that its small size could make it easier to work with in certain applications.
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