Megaphages harbor mini-Cas proteins ideal for gene editing
Hypercompact Cas protein should allow easier viral delivery of gene
editors

Date:
July 16, 2020
Source:
University of California - Berkeley
Summary:
Cas proteins like CRISPR-Cas9 have great potential for gene
therapy to treat human disease and for altering crop genes,
but the gene-targeting and gene-cutting Cas proteins are often
large and hard to ferry into cells with viral vectors such as
adenovirus. Scientists have now discovered a hypercompact Cas
protein, Cas-phi, that should work better.

It is half the size of Cas9 and apparently evolved inside a
bacteriophage, yet efficiently snips double-stranded DNA.



FULL STORY ==========================================================================
The DNA-cutting proteins central to CRISPR-Cas9 and related gene-editing
tools originally came from bacteria, but a newfound variety of Cas
proteins apparently evolved in viruses that infect bacteria.


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The new Cas proteins were found in the largest known bacteria-infecting viruses, called bacteriophages, and are the most compact working Cas
variants yet discovered -- half the size of today's workhorse, Cas9.

Smaller and more compact Cas proteins are easier to ferry into cells
to do genome editing, since they can be packed into small delivery
vehicles, including one of the most popular: a deactivated virus called adeno-associated virus (AAV). Hypercompact Cas proteins also leave space
inside AAV for additional cargo.

As one of the smallest Cas proteins known to date, the newly discovered
CasF (Cas-phi) has advantages over current genome-editing tools when
they must be delivered into cells to manipulate crop genes or cure
human disease.

"Adenoviruses are the perfect Trojan horse for delivering gene editors:
You can easily program the viruses to reach almost any part in the
body," said Patrick Pausch, a postdoctoral fellow at the University
of California, Berkeley, and in UC Berkeley's Innovative Genomics
Institute (IGI), a joint UC Berkeley/UCSF research group devoted to
discovering and studying novel tools for gene editing in agriculture
and human diseases. "But you can only pack a really small Cas9 into
such a virus to deliver it. If you would have other CRISPR-Cas systems
that are really compact, compared to Cas9, that gives you enough space
for additional elements: different proteins fused to the Cas protein,
DNA repair templates or other factors that regulate the Cas protein
and control the gene editing outcome." Apparently these "megaphages"
use the CasF protein -- the Greek letter F, or phi, is used as shorthand
for bacteriophages -- to trick bacteria into fighting off rival viruses, instead of itself.



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"The thing that actually made me interested in studying this protein specifically is that all the known CRISPR-Cas systems were originally discovered in bacteria and Archaea to fend off viruses, but this was
the only time where a completely new type of CRISPR-Cas system was first
found, and so far only found, in viral genomes," said Basem Al-Shayeb,
a doctoral student in the IGI. "That made us think about what could be different about this protein, and with that came a lot of interesting properties that we then found in the lab." Among these properties: CasF evolved to be streamlined, combining several functions in one protein,
so that it can dispense with half the protein segments of Cas9. It is as selective in targeting specific regions of DNA as the original Cas9 enzyme
from bacteria, and just as efficient, and it works in bacteria, animal
and plants cells, making it a promising, broadly applicable gene editor.

"This study shows that this virus-encoded CRISPR-Cas protein is actually
very good at what it does, but it is a lot smaller, about half the size
of Cas9," said IGI executive director Jennifer Doudna, a UC Berkeley
professor of molecular and cell biology and of chemistry and a Howard
Hughes Medical Institute investigator. "That matters, because it might
make it a lot easier to deliver it into cells than what we are finding
with Cas9. When we think about how CRISPR will be applied in the future,
that is really one of the most important bottlenecks to the field right
now: delivery. We think this very tiny virus-encoded CRISPR-Cas system
may be one way to break through that barrier." Pausch and Al-Shayeb are
first authors of a paper describing CasF that will appear this week in
the journal Science.

Biggiephages carry their own Cas proteins The CasF protein was first
discovered last year by Al-Shayeb in the laboratory of Jill Banfield,
a a UC Berkeley professor of earth and planetary science and environment science, policy and management. The megaphages containing CasF were
part of a group they dubbed Biggiephage and were found in a variety of environments, from vernal pools and water-saturated forest floors to
cow manure lagoons.



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"We use metagenomic sequencing to discover the Bacteria, Archaea and
viruses in many different environments and then explore their gene
inventories to understand how the organisms function independently and in combination within their communities," Banfield said. "CRISPR-Cas systems
on phage are a particularly interesting aspect of the interplay between
viruses and their hosts." While metagenomics allowed the researchers to isolate the gene coding for CasF, its sequence told them only that it
was a Cas protein in the Type V family, though evolutionarily distant
from other Type V Cas proteins, such as Cas12a, CasX (Cas12e) and
Cas14. They had no idea whether it was functional as an immune system
against foreign DNA. The current study showed that, similar to Cas9,
CasF targets and cleaves foreign genomes in bacterial cells, as well as double-stranded DNA in human embryonic kidney cells and cells of the
plant Arabidopsis thaliana. It also can target a broader range of DNA
sequences than can Cas9.

The ability of CasF to cut double-stranded DNA is a big plus. All other
compact Cas proteins preferentially cut single-stranded DNA. So, while
they may fit neatly into compact delivery systems like AAV, they are
much less useful when editing DNA, which is double-stranded, inside cells.

As was the case after Cas9's gene-editing prowess was first recognized
in 2012, there is a lot of room for optimizing CasF for gene editing and discovering the best rules for designing guide RNAs to target specific
genes, Pausch said.

Other co-authors of the paper are Ezra Bisom-Rapp, Connor Tsuchida, Brady
Cress and Gavin Knott of UC Berkeley and Zheng Li and Steven E. Jacobsen
of UCLA. The researchers were funded, in part, by the Paul G. Allen
Frontiers Group, National Institutes of Health Somatic Cell Genome Editing consortium (U01AI142817-02) and National Science Foundation (DGE 1752814).


========================================================================== Story Source: Materials provided by
University_of_California_-_Berkeley. Note: Content may be edited for
style and length.


========================================================================== Journal Reference:
1. Patrick Pausch, Basem Al-Shayeb, Ezra Bisom-Rapp, Connor
A. Tsuchida,
Zheng Li, Brady F. Cress, Gavin J. Knott, Steven E. Jacobsen,
Jillian F.

Banfield, Jennifer A. Doudna. CRISPR-CasF from huge
phages is a hypercompact genome editor. Science, 2020 DOI:
10.1126/science.abb1400 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200716144728.htm

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