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About a decade ago, scientists discovered the function of CRISPR, a tool that bacteria use to protect themselves from viruses
"However, the Cas9 tool does have limited capabilities for large-scale gene editing," said Yan Zhang , Ph.
That's where another editing tool, CRISPR-Cas3, comes in
In 2019, Zhang's lab was one of the first researchers to describe the new tool, which has the ability to degrade DNA, resulting in large-scale genome deletions
However, it faces some limitations in efficiency, larger gene size and protein manufacturing, Zhang said
In the new paper, they show the first example of a miniature CRISPR-Cas3 editor extracted from the bacterium Neisseria lactamica , which greatly improves editing efficiency and is easier to use as a ribonucleoprotein reagent production, and have a more compact overall gene size that facilitates in vivo delivery
The team has now developed the Neisseria lactamica CRISPR-Cas3 purified Cascade RNP and Cas3 protein, which can achieve 50% editing efficiency in stem cells and 95% editing efficiency in other human cell lines
However, when they tried to make and deliver the plasmid, they didn't work
PhD student Renke Tan noticed an unexpected protein band on the protein purification gel and eventually discovered that it was an internal translation product, Cas11, without which the CRISPR-Cas3 complex would not have formed, and therefore would not have been observed in humans.
"This type of Cas11 protein is like a hidden gem that we and other researchers have not discovered until recently," Zhang said
Adding it back via a separate expression plasmid produces the Cas11 protein in human cells and allows the CRISPR-Cas3 plasmid system to work properly
With the publication of this work, the team has made the tool available on Addgene for researchers to investigate a wide range of scientific questions, from deleting large disease-causing genes to studying the noncoding human genome
The University of Michigan and Cornell University have filed a joint patent on the discovery
“Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems,” Molecular Cell .
