The ability to label and manipulate proteins
in the body is essential to modern biological
research.
Unfortunately, current methods, such as tagging
with antibodies, are often inefficient and
expensive.
Even worse, researchers are realizing that
many of the antibodies available just simply
don’t work. Now, a new molecular tool could
help researchers break through that barrier.
Researchers in the Soderling Laboratory of
the Cell Biology Department at Duke University, have
developed a high-throughput system capable
of modifying entire panels of proteins using
a new dual-vector gene-editing approach.
Dubbed Homology-independent Universal Genome
Engineering, this system allows for the dynamic
visualization and functional manipulation
of proteins both in vitro and in vivo, including
in neurons. This is HiUGE.
HiUGE isn’t the first protein-modifying
system to rely on gene editing. Techniques
such as single-cell labeling of endogenous
proteins (SLENDR) or homology-independent
targeted integration (HITI) have made it possible
to insert foreign DNA sequences into genes
of interest.
The difference: these methods require customized
gene-specific donor vectors for each insertion;
HiUGE doesn’t.
In HiUGE, the donor vector contains an insertional
DNA payload flanked by an artificial DNA sequence
non-homologous to the target genome.
This sequence is recognized by a donor-specific
guide RNA that autonomously directs Cas9-mediated
payload clipping and release.
Separate, gene-specific gRNA vectors then
designate the payload target in the gene of
interest.
This design frees the donor vectors of any
gene-specific sequences.
The result is the potential to create high-throughput
donor “toolkits” that target a variety
of genes rather than just one.
In addition, HiUGE employs adeno-associated
virus as an efficient vehicle to deliver these
"toolkits" to cells or even tissues in animals. As
a proof of concept, the research team co-transduced
primary neurons from neonatal mouse pups with
two sets of vectors: one containing gRNA targeting
the mouse Tubb3 gene and the other containing
the machinery to insert the protein tag hemagglutinin,
or HA.
After about one week, fluorescence detection
revealed successful HA labeling.
Genomic insertion of the payload was verified
by sequencing the Tubb3 locus.
In further tests, HiUGE proved capable of
targeting multiple genomic loci for protein
labeling, labeling proteins in vivo, delivering
different payloads interchangeably at a single
genomic locus, and targeting specific neural
circuits. Potential drawbacks of all CRISPR-dependent
systems, including HiUGE, is the formation
of indels at the targeted loci or off-target
insertion of genomic payloads.The team found
with careful design these effects can be greatly
minimized.
And the benefits are very promising.
Scalable, efficient, and universally compatible
for virtually any loci accessible by CRISPR/Cas9,
HiUGE opens the door to pairing high-throughput
“omics” with experimental validation and
phenotypic screening to address molecular
mechanisms of cellular biology.
