RESUMO
The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021).
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/efeitos da radiação , Células HEK293 , Humanos , Mutação INDEL/genética , Fotólise , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Guia de Cinetoplastídeos/efeitos da radiaçãoRESUMO
The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.
Assuntos
Proteína 9 Associada à CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/efeitos da radiação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Cultura de Células , Reparo do DNA por Junção de Extremidades , Células HEK293 , Humanos , Mutação INDEL , Mutação Puntual , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.
