The CRISPR-Cas9 genome editing system has
revolutionized biomedical science, providing
a fast and easy way to modify genes.
The version of the technique that allows for
the most precise edits, though, doesn’t
work in cells that are no longer dividing.
Since that includes most of the neurons in
the brain, this has limited the technology
for neuroscientists.
But now, a group at the Max Planck Florida
Institute for Neuroscience has figured out
a way to make CRISPR work in these cells,
opening up new possibilities for the field.
CRISPR-based editing uses a guide RNA to direct
the cas9 endonuclease to a specific spot in
the genome and make a cut in the DNA.
Cellular repair mechanisms then kick into
action, either in the form of non-homologous
end joining, which causes unpredictable insertions
or deletions, or with homology-directed repair,
which uses a donor template to make a precise
change.
Unfortunately, because homology-directed repair
has been thought to only happen in the S and
G2 phases of the cell cycle, this more desirable
method does not work in postmitotic cells,
such as neurons in the brain.
To overcome this problem, neuroscientists
added adeno-associated virus, or AAV, to the
mix.
This virus can effectively provide the donor
template necessary for homology-directed repair.
Therefore, it seems to increase gene targeting.
The team tested the approach in mouse brain
slices, using CRISPR to add HA or GFP to a
protein found in neurons.
The gene editing beautifully lit up neurons
-- many of which were no longer dividing -- in
genetically engineered mice expressing the
cas9 protein.
But the success rate depended on the dose
of the virus.
To more carefully study this phenomenon, the
scientists set up cultures of mitotic and
postmitotic hippocampal cells, and found that
postmitotic cells need about 100 times more
virus to get CRISPR to work.
The group then created a dual-viral system
so they could use the technology in many animals
that have not been engineered to express cas9.
This worked in both rat and mouse brain slices.
Finally, the team tested the dual-viral system
in living mice, including an aged Alzheimer’s
disease mouse model by directly injecting
AAVs into the brain.
The new method, which the team calls vSLENDR,
is capable of working in virtually any brain
area, cell type, and age -- regardless of
whether cell division is still happening -- greatly
expanding the types of experiments neuroscientists
can do to probe the function of the brain.
