Hidden Protein Essential for CRISPR-Cas3 Efficiency

A new gene-editing tool is enabling CRISPR technology to make larger edits with high efficiency, overcoming a key limitation to the use of the technology in genetic disease and developmental biology research.

Developed nearly a decade ago, CRISPR-Cas9 has become a workhorse for genome editing, allowing researchers to identify and replace targeted DNA within a cell. However, the ability of cas9 tools to make larger edits is limited. More recently, CRISPR-Cas3 was developed. Through processive DNA degradation, newer tool Cas3 can make larger deletions on the order of the average human gene (10-15 kilobases), but it had limitations of its own, namely around efficiency, larger gene size and protein manufacturing.

In a paper published recently in the journal Molecular Cell, Yan Zhang, Ph.D., assistant professor in the Department of Biological Chemistry at the University of Michigan Medical School, and colleagues describe a miniature CRISPR-Cas3 editor from the bacteria Neisseria lactamica that greatly improves editing efficiency, is easier to produce, and has a more compact overall gene size that favors in vivo delivery.  

The breakthrough came when the team set out to establish an expression method using plasmids, the small circular DNA molecules that researchers use to circumvent the protein purification step. When they attempted to make and deliver the plasmids, they didn’t work. That was when Ph.D. student and coauthor Renke Tan noticed an extra, unexpected protein band on the protein purification gel, and eventually figured out that it was internal translation product Cas11, without which the CRISPR-Cas3 complex would not form.

“This type of Cas11 protein was like a hidden gem that’s been only recently noticed by us and other researchers,” said Zhang. Adding it back through a separate expression plasmid enabled the production of Cas11 protein in human cells and allowed the CRISPR-Cas3 plasmid system to function robustly.

Using the older version of the tool, the team achieved only 10% editing in stem cells and 30-50% in cancer-derived cell lines. According to the paper, using the N. lactamica CRISPR-Cas3, the team was able to achieve 50% editing efficiency in stem cells and an 95% efficiency in other human cell lines.

“I think we found the version of CRISPR-Cas3 tool that we are very satisfied with,” said Zhang.

The team has made the tool available on Addgene, and U-M and Cornell have filed for a joint patent.