A detailed protocol for expression, purification, and activity determination of recombinant SaCas9

Recombinant SaCas9 is useful for a broad range of applications in the context of genome editing, especially when the specific protospacer adjacent motifs of other Cas9 derivatives are missing. Here, we describe a detailed protocol for the expression and purification of recombinant SaCas9. We detail...

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Detalles Bibliográficos
Autores principales: Flottmann, Franziska, Pohl, Greta Marie, Gummert, Jan, Milting, Hendrik, Brodehl, Andreas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8991255/
https://www.ncbi.nlm.nih.gov/pubmed/35403007
http://dx.doi.org/10.1016/j.xpro.2022.101276
Descripción
Sumario:Recombinant SaCas9 is useful for a broad range of applications in the context of genome editing, especially when the specific protospacer adjacent motifs of other Cas9 derivatives are missing. Here, we describe a detailed protocol for the expression and purification of recombinant SaCas9. We detail the main steps for immobilized metal affinity chromatography and size exclusion chromatography. In addition, we present an assay for activity determination of SaCas9. Active SaCas9 can be purified in a week by using this protocol.

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