Breakthrough Genome-editing Technologies
Demeetra AgBio, Inc. gene-editing technologies are broken down into two categories, including, piggyBac transposase and Cas-CLOVER. Each technology has different mechanisms, advantages, and uses and they can be used together for Footprint-Free gene editing. Demeetra utilizes these technologies for its own development pipeline and partners with researchers through licensing or collaborations in the Agriculture, Industrial Biotechnology & Pharmaceutical Bioprocessing industries.
Our Proprietary Genome Editing Toolbox
“Molecular Scissors” introduce targeted double-strand breaks in genomic DNA with knockouts, knock-ins and base-pair edits.
Introduction of small to very large genetic cargo and removal of genetic cargo in a scarless manner in transgenics and stable cell lines.
What is Cas-CLOVER
Hybrid gene editing system using a nuclease-inactivated Cas9 protein fused to a Clo51 endonuclease.
dCas9 serves as a fusion protein only. It is mutated and unable to cut DNA.
Cleavage activity depends on the dimerization of an “obligate dimer” of Clo51.
Benfits:
Ease of use: double gRNA guided
High efficiency: dCas9-guided RNA recognition
High fidelity: cuts only when the Clo51 nuclease dimerizes
How does PiggyBac Transposase Work
Step 1: Clone cargo into transposon vector.
Step 2: Co-transfect transposon and transposase into cells.
Step 3: Transposase cuts out cargo and pastes into random TTAA sites.
Step 4: Screen clones for desired insert.
Step 5 (optional): Use excision-only piggyBac for footprint-free gene editing.
Features:
Combining Cas-CLOVER and PiggyBac for Footprint-Free Gene Editing
Step 1: With Cas-CLOVER, generate a double stranded break in the genomic DNA next to the desired gene edit. Using homologous recombination (HR) insert the desired gene edit along with a positive/negative selectable marker flanked by the piggyBac target sequences, inverted terminal repeats (ITRs).
Step 2: Select for the targeted integration of your desired gene edit, increasing efficiency of a sometimes very rare event by killing off all non-edited cells.
Step 3: Introduce excision-only piggyBac transposase which seamlessly removes the selectable marker leaving behind a correctly edited cell with no other footprints.
Footprint-Free gene editing version 1
Footprint-Free gene editing version 2
Step 1: Stably introduce Cas-CLOVER into the cell or organism of choice using piggyBac transposase.
Step 2: The stable integration of Cas-CLOVER increases expression and editing efficiency.
Step 3: Introduce the gRNA and/or donor vectors for gene editing.
Step 4: Excision-only piggyBac is used to remove stably Cas-CLOVER, leaving behind the desired edit without a trace.