AI Article Synopsis
- Researchers have created a new gene delivery tool called FiCAT that combines CRISPR-Cas9 precision with a modified piggyBac transposase for better integration of large DNA fragments into mammalian genomes.
- FiCAT enhances the targeting of DNA and improves the transfer of donor DNA, increasing the accuracy of genetic modifications and decreasing unintended effects.
- The tool has been tested successfully in various human and mouse cell lines, and through extensive experimentation, improved versions of FiCAT were developed, enabling efficient insertion of large DNA sequences in living organisms.
Article Abstract
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8642419 | PMC |
| http://dx.doi.org/10.1038/s41467-021-27183-x | DOI Listing |
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