While tools to manipulate viral nucleic acids have improved rapidly, methods to rescue infectious virus from
genomic material that is not infectious have not. Methods for virus rescue are available for increasing numbers
of virus species but must be individually developed and optimized for each different group of related viruses—a
process that is time-consuming and costly. Therefore, there is a need to develop a generalizable synthetic
genomics technology to facilitate rapid rescue of broad range of viruses at a reduced research cost to speed the
development of genetic tools for novel, emerging, or understudied viruses. Here, we propose to develop FRESH
(Fast Rescue Employing Self-Helper virus), a rescue strategy for viruses with noninfectious genomes and test
its applicability for rescue of DNA and RNA viruses in prokaryotic and eukaryotic hosts. To boot infectivity of the
genomic material with FRESH, a helper virus of the same virus species supplies trans-acting factors. In order to
inhibit packaging of the helper virus, its genome is differentially targeted for CRISPR-Cas digestion. Sequence
variation between the helper virus genome and the rescued genome, due to existing natural inter-strain variation
or engineered mutations, confer immunity to the CRISPR guide RNAs. We have used FRESH to rescue African
swine fever virus, which has a large DNA genome that is not infectious and no reported rescue methodology. To
test the effectiveness of FRESH in a bacterial host, we propose to rescue P68, a virus of Staphylococcus, and
inhibit the helper virus strain with CRISPR-Cas9 digestion. We will also develop FRESH with a Cas13 effector
in mammalian cells to rescue human respiratory syncytial virus (RSV), as a model for negative-sense single-
stranded RNA viruses. For each virus, we will determine the most effective guide RNAs, which will be used to
inhibit the wild-type self-helper virus. Since we have already developed synthetic genomics tools for P68 and
RSV, we will engineer the genomes to include a fluorescent reporter and to modify the sgRNA target regions,
thus generating a CRISPR-immune reporter genome to be rescued. If these experiments are successful,
additional work will be performed to define a straightforward FRESH rescue system that could be rapidly
employed for previously undescribed viruses. Combined, FRESH and synthetic genomics can rapidly introduce
sophisticated reverse genetics to virus species with noninfectious genomes allowing researchers to begin
developing effective viral countermeasures in less time.
