Purification and functional validation of LtCas12a protein

• Published in: STAR protocols Vol. 4; no. 4; p. 102600
• Main Authors: Zhou, Bo, Chen, Ye, Li, Lifang, Liu, Jiashuo, Wang, Yuyan, Huang, Zheying, Hu, Zheng, Tian, Rui, Li, Zhen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.12.2023; Elsevier
• Subjects: Cancer; Chromatography, Affinity; CRISPR; Genetics; Protein Biochemistry; Protein Expression and Purification; Protocol; Structural Biology; CRISPR; Genetics; Protein Expression and Purification; Protein Biochemistry; Structural Biology; Cancer
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102600

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 [Display omitted] •Detailed protocol for a Cas12a ortholog with TTNA PAM•Purification of LtCas12a via multiple chromatographic techniques•Validation of LtCas12a biological characteristics by various in vitro assays Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.