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
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author Zhou, Bo
Chen, Ye
Li, Lifang
Liu, Jiashuo
Wang, Yuyan
Huang, Zheying
Hu, Zheng
Tian, Rui
Li, Zhen
author_facet Zhou, Bo
Chen, Ye
Li, Lifang
Liu, Jiashuo
Wang, Yuyan
Huang, Zheying
Hu, Zheng
Tian, Rui
Li, Zhen
author_sort Zhou, Bo
collection PubMed
description 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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spelling pubmed-105508372023-10-06 Purification and functional validation of LtCas12a protein Zhou, Bo Chen, Ye Li, Lifang Liu, Jiashuo Wang, Yuyan Huang, Zheying Hu, Zheng Tian, Rui Li, Zhen STAR Protoc Protocol 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) Elsevier 2023-09-27 /pmc/articles/PMC10550837/ /pubmed/37768827 http://dx.doi.org/10.1016/j.xpro.2023.102600 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Zhou, Bo
Chen, Ye
Li, Lifang
Liu, Jiashuo
Wang, Yuyan
Huang, Zheying
Hu, Zheng
Tian, Rui
Li, Zhen
Purification and functional validation of LtCas12a protein
title Purification and functional validation of LtCas12a protein
title_full Purification and functional validation of LtCas12a protein
title_fullStr Purification and functional validation of LtCas12a protein
title_full_unstemmed Purification and functional validation of LtCas12a protein
title_short Purification and functional validation of LtCas12a protein
title_sort purification and functional validation of ltcas12a protein
topic Protocol
url 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
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