RESUMO
TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.
Assuntos
Marcação de Genes/métodos , Genoma/genética , Ativação Transcricional/genética , Transfecção/métodos , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células HCT116 , Humanos , Dados de Sequência MolecularRESUMO
Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities. We demonstrate the modularity of the chimeric DmoCre meganuclease previously described, by successfully assembling mutants with locally altered specificities affecting both I-DmoI and I-CreI subdomains in order to create active meganucleases with altered specificities. Moreover these new engineered DmoCre variants appear highly specific and present a low toxicity level, similar to I-SceI, and can induce efficient homologous recombination events in mammalian cells. The DmoCre based meganucleases can therefore offer new possibilities for various genome engineering applications.
Assuntos
Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Animais , Células CHO , Sobrevivência Celular , Técnicas de Química Combinatória , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Mutagênese , Mutação , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato/genéticaRESUMO
The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.