Find and cut-and-transfer (FiCAT) mammalian genome engineering
| Autor: | Dimitrije Ivančić, Jessica Jaraba-Wallace, Amal Rahmeh, Tommaso Tagliani, Baldomero Oliva, Maria Pallarès-Masmitjà, Marc Güell, Júlia Mir-Pedrol, Avencia Sánchez-Mejías |
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| Rok vydání: | 2021 |
| Předmět: |
Male
Transfer capacity Computer science Science Transposases General Physics and Astronomy Computational biology Protein Engineering Genome Article General Biochemistry Genetics and Molecular Biology Cell Line Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing Animals Humans Gene Transposase 030304 developmental biology Gene Editing 0303 health sciences Multidisciplinary Cas9 General Chemistry Targeted gene repair Electroporation Liver chemistry Genetic engineering DNA Transposable Elements Female Mammalian genome CRISPR-Cas Systems 030217 neurology & neurosurgery DNA |
| Zdroj: | Nature Communications Nature Communications, Vol 12, Iss 1, Pp 1-9 (2021) |
| ISSN: | 2041-1723 |
| Popis: | 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. Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models. |
| Databáze: | OpenAIRE |
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