Plug-and-play protein modification using Homology-independent Universal Genome Engineering (HiUGE)
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Plug-and-play protein modification using Homology-independent Universal Genome Engineering (HiUGE)

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.

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