Dr. Pablo Perez-Pinera from the University of Illinois has developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. Furthermore, this method facilitates activation of genes by introduction of specific promoters that allows for genomic scale activation screens.
Benefits
• Minimizes cost and time for gene editing • Multigene knockouts can be rapidly generated • Facile integration of large constructs up to 50 kb • Introduces speci c promoters for genomic scale activation screens
Dr. Pablo Perez-Pinera has developed a method to divide prime editing tools into smaller components that are small enough to be delivered in adeno-associated viruses, an...
Dr. Pablo Perez-Pinera has developed a method to divide prime editing tools into smaller components that are small enough to be delivered in adeno-associated viruses, an FDA-approved vehicle for therapeutic use. Prime editors have recently shown great promise as gene editing tools with greater specificity and fewer off-target effects than existing CRISPR-Cas based methods. This technology has potential as a research tool as well as a therapeutic strategy.
Dr. Thomas Gaj has developed a novel prime editor based on a Staphylococcus aureus Cas9 ortholog. Furthermore, he has developed split intein versions of the prime editing...
Dr. Thomas Gaj has developed a novel prime editor based on a Staphylococcus aureus Cas9 ortholog. Furthermore, he has developed split intein versions of the prime editing tools that are small enough to be dual-delivered in adeno-associated viruses, an FDA-approved vehicle for therapeutic use. This invention has been shown to improve targeting capability of the prime editors by several fold. Prime editors have recently shown great promise as gene editing tools with greater specificity and fewer off-target effects than existing CRISPR-Cas based methods. This technology has potential as a research tool as well as a therapeutic strategy.
Genetic engineering often uses protein enzymes for sequence-specific nucleic acid manipulation, but these enzymes lack customizability. Programmable systems like TALEN and...
Genetic engineering often uses protein enzymes for sequence-specific nucleic acid manipulation, but these enzymes lack customizability. Programmable systems like TALEN and CRISPR/Cas address this but have limitations, such as engineering difficulties and off-target effects. Large protein enzymes may also struggle to access tightly packed chromatin. Catalytic nucleic acid-based systems, like DNAzymes, are smaller, more stable, and cost-effective, and have been used in various applications. However, their activity on double-stranded DNA (dsDNA) has not been verified, limiting their use in direct gene manipulation.
This is a method for genome editing of double-stranded DNA with higher specificity than existing genome editing technologies. As opposed to relying on a site-specific guide RNA and a non-specific enzyme with the CRISPR/Cas9 system, this method uses Peptide Nucleic Acid (PNA) and DNAzyme, where both components are site specific. The PNA binds to a single strand of dsDNA for the DNAzyme to target and cleave the second strand of DNA. This system achieves the optimal conditions necessary for both PNA invasion of the dsDNA and the DNAzyme cleavage activity. With modification to the conditions, the PNA in this system can be replaced with long homologous ssDNA for increased cost-effectiveness. Inventors have also shown the use of an interstrand zipper for dsDNA invasion, instead of PNA (Tech ID 2024-242).
Publication
PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity. Mingkuan Lyu, Linggen Kong, Zhenglin Yang, Yuting Wu, Claire E. McGhee, and Yi Lu. Journal of the American Chemical Society 2021 143 (26), 9724-9728 DOI: 10.1021/jacs.1c03129
