Purification and functional validation of LtCas12a protein

Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We t...

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Detalles Bibliográficos
Autores principales: Zhou, Bo, Chen, Ye, Li, Lifang, Liu, Jiashuo, Wang, Yuyan, Huang, Zheying, Hu, Zheng, Tian, Rui, Li, Zhen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10550837/
https://www.ncbi.nlm.nih.gov/pubmed/37768827
http://dx.doi.org/10.1016/j.xpro.2023.102600
Descripción
Sumario:Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We then detail procedures for verification of LtCas12a with cis- and trans-cleavage activities. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023).(1)

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