Each type of CRISPR has its own unique and special properties that allow it to edit and modify DNA, and in some cases RNA, in a variety of useful manners. This includes Cas10’s ability to target mutated sequences, Cas1 and Cas2’s viral DNA insertion abilities, and Cas12’s high detection abilities for viral DNA hiding in complicated structures, as we’ve discussed before.

But while all of that is well and good, it is the fundamental ability of CRISPR to insert desired target DNA into a cell that has the biggest benefit for medical and biological science and is why Cas9 has become such a ubiquitous tool throughout the world. It is, however, still very specific in what it does and we love it for that. You give it a sequence and it goes to that sequence to cut out or insert the DNA at that point. Just like you want it to.

Back To The Older CRISPR

What if, though, you want to have a genome-wide change that is able to target common sequences found across the genome and yet is still able to precisely go after single gene size sequences? Well, then you’re going to want an alternative CRISPR complex known as Cas3. That tool was first described by the University of Michigan’s Zhang lab back in 2019 and was highly praised for its capability to create multiple deletions in an entire genome. But there were some limitations. 

These Type I forms of CRISPR, as briefly discussed in our primer, were dubbed Cascade thanks to the cascading activation system they used to respond to viral invasion. And while highly effective, they have a size issue. 

A large number of cas genes make up the Cascade and Cas3 complexes, making them on average more than 50% larger than a comparative Cas9 complex. This has resulted in Cas3 only really being tested and used in bacterial systems and not on eukaryotic cells, as the manner of getting the complex into those types of cells would be prohibitively difficult. 

Bringing In New From The Old

So the Zhang lab decided to go back to the bacteria themselves and find a Cas3 system that can be repurposed into a eukaryotic cell tool. And they found just what they wanted in the bacteria Neisseria lactamica that contains a miniaturized Cas3 system that still has a highly active functionality. 

While testing its usage in eukaryotic cells, they also made a discovery about another Cas component that was critical to Cas3’s activity. Cas11, which is usually just a smaller component of Cas8 and involved in gene translation, but without it, no editing of the genome occurs despite confirmed expression of the other genes. The internal translation mechanism of Cas11 is needed and can also be independently added into Cas3 systems from other bacterial sources to further improve their editing capabilities. In total, the newly improved tool is able to have an editing efficiency improvement from less than 50% with the older system to 95% with this new one, also increasing the efficiency of editing stem cells from 10% to 50%. 

A Whole Genome of Modification

The researchers also hope that this discovery of how Cas11 is involved will allow it to be used to further improve the efficiencies of other Cas systems throughout the Type I CRISPR lineage. They have openly allowed access to the tool for any scientist to use and will be using the tool themselves in future animal models for controlling and testing diseases and the ability of differentiated cells to be reversed into stem cell-like forms through modifying their genetics.

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Photo CCs: APAF 1 from Wikimedia Commons

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