Our Solution

Our Proposed Solution 

For our design, we will test and compare the efficacy of three different Cas systems, dCas9, dCpf1, and dCasMini, paired with the activator domain VPR. Initially, we will experiment with integrating gRNAs targeting NEUROG2, a key transcription factor in neuron differentiation. Previous experiments with direct overexpression of this transcription factor have shown rapid conversion of neurons from stem cells. Once we confirm that this system can generate neurons, we will begin multiplexing and testing different combinations of gRNAs targeting other transcription factors to generate different retinal neuron subtypes. Downstream applications of our system include in vivo adeno-associated virus (AAV) gene therapy in 3D retina organoid models. Eventually, we will consider FDA standards for off-target effects, efficacy, and consistency for our solution in therapeutic contexts.

To do so, we cloned two different types of plasmids. Our activator plasmid (top) carries the sequence for the dCas protein in each system we are exploring. Some important elements of this plasmid include:

• Tet-inducible promoter, meaning that the system's activation can be precisely controlled using doxycycline 

• AAVS1 homology arms to ensure integration of the plasmid into well-characterized safe harbor sites

• mRuby fluorescent marker to validate transfection and integration of the plasmids in the stem cells using a fluorescence microscope.

• Hygromycin selectable marker to enable purification of the cell lines.

The gRNA plasmid (bottom) contains gRNA sequences that will guide the dCas-VPR complex (once translated from the activator plasmid) to the transcription factor of interest. We cloned gRNA plasmids for two transcription factors - NEUROG2 and ASCL1 - important for retinal development. Several gRNA sequences targeting each transcription factor were sourced and cloned, as sourced from scientific literature. Like the activator plasmid, the gRNA plasmid contains a puromycin selectable marker and ROGI1 homology arms to enable downstream purification and integration into a safe harbor site. 

Alternative Solution

PiggyBac transposon is an innovative technique for integrating transgenes into DNA using transposase enzymes. These enzymes cut the target DNA from the donor site at specific sequences known as inverted terminal repeats (ITRs). Subsequently, the transposase enzymes, now in a dimerized structure referred to as the "transposome," facilitate the relocation of the transgene to a TTAA site within the host genome.1 The TTAA site is particularly noteworthy in the context of stem cell differentiation into retinal neurons. This site is often found in open chromatin regions, making it easily accessible to transcription factors. This accessibility, in turn, enhances the expression of the integrated transgene, a crucial factor in guiding stem cells toward a retinal neuron fate. Importantly, the one-to-one binding of a dimerized transposome to a TTAA site ensures a consistent and reliable expression of the transgene in the differentiated retinal neurons. This makes it efficient for achieving the desired cellular outcomes in retinal neuron differentiation from stem cells.

Because we could not successfully integrate the activator construct into the AAVS1 safe harbor site due to epigenetic silencing, we switched out the homology arms in the activator plasmid for the TTAA site recognized by transposase. Transposase will recognize these ITRs and semi-randomly insert several copies of the construct into the genome. Ideally, this should overcome the problem of epigenetic silencing.