Prokaryotic Expression, Purification of LbCpf1 Protein Gene and in Vitro Cleavage Activity Assay

doi:10.13523/j.cb.2003057

China Biotechnology

2020, Vol. 40

Issue (8): 41-48 DOI: 10.13523/j.cb.2003057

Prokaryotic Expression, Purification of LbCpf1 Protein Gene and in Vitro Cleavage Activity Assay

LV Yi-fan,LI Geng-dong,XUE Nan,LV Guo-liang,SHI Shao-hui,WANG Chun-sheng(

)

College of Life Science, Northeast Forestry University, Harbin 150040, China

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Abstract

Compared to Cas9, LbCpf1 has higher targeting specificity and other advantages in eukaryotic cells. Therefore, this study aims to obtain the LbCpf1 protein that is cleaved by in vitro activity. To achieve that, pY016 plasmids containing LbCpf1 gene coding region of Lachnospiraceae bacterium ND2006 were double-enzyme digested to obtain the CRISPR-LbCpf1 gene CDS. Next, the prokaryotic expression plasmid CRISPR-LbCpf1-6*His was constructed by ligating the CRISPR-LbCpf1 gene sequence to the prokaryotic expression vector pHis*6(IV) containing the 6*His tag. Afterwards, high yields of recombinant plasmids were obtained from transformed DH5α competent cells. Then the obtained plasmids were identified by double-enzyme digestion and sequencing, the results of which showed the correct recombinant plasmids were constructed successfully. The correct plasmids were subsequently transformed into E. coli BL21 (DE3) competent cells to generate a BL21(DE3) expression strain containing the recombinant plasmid CRISPR-LbCpf1-6*His, which were then inoculated and cultivated at 37°C, on a 160 r/min shaker. The expression of target gene was induced by IPTG (final concentration 0.5 mmol/L) for 5 hours, and the production was purified by Ni column affinity chromatography, dialysis and desalting, SDS-PAGE gel electrophoresis and other steps to obtain the recombinant protein. The final concentration of the protein can reach approximately 400 ng/μl. Finally, via in vitro cleavage assay, it showed the protein was able to process the pre-crRNA in an appropriate environment and bind to the mature CRISPR RNA (crRNA) to cleave the target DNA in vitro, which verified the recombinant protein cleavage activity. In conclusion, this study provides a method to obtain high-purity LbCpf1 protein, supporting the usage of LbCpf1 in further genetic editing research.

Key words： LbCpf1 Prokaryotic expression Nickel column purification In vitro cleavage activity assay

Received: 23 March 2020 Published: 10 September 2020

ZTFLH:

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Corresponding Authors: Chun-sheng WANG E-mail: wangchunsheng79@nefu.edu.cn

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	Yi-fan LV
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	Shao-hui SHI
	Chun-sheng WANG

Cite this article:

LV Yi-fan,LI Geng-dong,XUE Nan,LV Guo-liang,SHI Shao-hui,WANG Chun-sheng. Prokaryotic Expression, Purification of LbCpf1 Protein Gene and in Vitro Cleavage Activity Assay. China Biotechnology, 2020, 40(8): 41-48.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2003057 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I8/41

Fig.1 Plasmid pHis*6 digested by Hind III and Xho I and plasmid map (a) Plasmid pHis*6 digested with Hind III and Xho I M: DNA DL 10 000 Maker; 1: Plasmid pHis*6 double digestion product (b) Plasmid map of pHis*6

Fig.2 Electrophoresis and plasmid map of plasmid pY016 double-digested product (a) Plasmid pY016 double digestion product electrophoresis 1: The production of double enzyme digestion for pY016 M: DNA DL 10 000 Marker; A: Plasmid pY016 digestion fragment; B: LbCpf1 destination fragment (b) Plasmid map of pY016

Table 1 crRNA sequence

Table 2 Target sequence PCR primer

Table 3 Mole ratio of LbCpf1, crRNA and DNA during in vitro cleavage

Fig.3 CRISPR-LbCpf1-6*His double-digested product electrophoresis and plasmid map (a) CRISPR-LbCpf1-6*His double enzyme digestion 1: Double-digested product of recombinant plasmid CRISPR-LbCpf1-6*His by HindⅢ and XhoⅠ. M: DNA Maker a: Plasmid pHis*6 vector; b: LbCpf1 gene fragment (b) CRISPR-LbCpf1-6*His plasmid map

Fig.4 SDS-PAGE analysis of IPTG-induced LbCpf1 M: Protein Marker; 1: 0.5mmol/L IPTG induction for 0 h; 2: 0.5mmol/L IPTG induction for 1h; 3: 0.5mmol/L IPTG induction for 2h; 4: 0.5mmol/L IPTG induction for 3h; 5: 0.5mmol/L IPTG induction for 4h; 6: 0.5mmol/L IPTG induction for 5h; 7: 0.5mmol/L IPTG induction for 6h; 8: 0.5mmol/L IPTG induction for 7h; 9: 0.5mmol/L IPTG induced lysate supernatant for 5h; 10: 0.5mmol/L IPTG induced precipitation for 5h

Fig.5 Analysis of expression types of targeted LbCpf1 recombinant proteins 1: Cell lysate supernatant after 5h induction by IPTG; 2: Precipitation of bacterial lysate after induction by IPTG for 5h; M: Protein Marker

Fig.6 Purification analysis of LbCpf1 target recombinant protein 1: Flow-through of supernatant; 2: 10mmol/L imidazole 4ml purified sample for the first time elution; 3: 10mmol/L imidazole 4ml second eluted purified sample; 4: 20mmol/L imidazole 4ml purified sample for the first time elution; 5: 20mmol/L imidazole 4ml second eluted purified sample; 6: 20mmol/L imidazole 4ml third eluted purified sample; 7: 50mmol/L imidazole 4ml purified sample for the first time elution; 8: 50mmol/L imidazole 4ml second eluted purified sample; 9: 50mmol/L imidazole 4ml third eluted purified sample; 10: 100 mmol/L imidazole 4ml purified sample for the first time elution. 11: 100mmol/L imidazole 4ml second eluted purified sample; 12: 200mmol/L imidazole 4ml purified sample for the first time elution;13: 200mmol/L imidazole 4ml second eluted purified sample

Fig.7 Agarose gel electrophoresis analysis of transcription templates and crRNA (a) Agarose gel electrophoresis analysis of transcription templates (b) Agarose gel electrophoresis analysis of crRNA

Fig.8 Schematic illustration of target sequence electrophoresis and binding of crRNA to target sequence (a) Target sequence gel electrophoresis analysis (b) Schematic diagram of crRNA binding to target sequence

Fig.9 In vitro cleavage detection of LbCpf1 protein (a)In vitro cleavage detection of recombinant LbCpf1 protein 1: Final concentration of 423 ng of protein LbCpf1; 2: Final concentration of 282 ng of protein LbCpf1; 3: No protein added (b) LbCpf1 protein produced by a company


[1]	Zetsche B, Gootenberg J S, Abudayyeh O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015,163(3):759-771. doi: 10.1016/j.cell.2015.09.038 pmid: 26422227

[2]	Gao P, Yang H, Rajashankar K R, et al. Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Res, 2016,26(8):901-913. doi: 10.1038/cr.2016.88 pmid: 27444870

[3]	Fonfara I, Richter H, Bratovič M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA . Nature, 2016,532(7600):517-521. doi: 10.1038/nature17945 pmid: 27096362

[4]	Zetsche B, Heidenreich M, Mohanraju P, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol, 2017,35(1):31-34. doi: 10.1038/nbt.3737 pmid: 27918548

[5]	Bin Moon S, Lee J M, Kang J G, et al. Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3'-overhang. Nat Commun, 2018,9(1):3651. doi: 10.1038/s41467-018-06129-w pmid: 30194297

[6]	Park H M, Liu H, Wu J, et al. Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo. Nat Commun, 2018,9(1):3313. doi: 10.1038/s41467-018-05641-3 pmid: 30120228

[7]	Wu H, Liu Q, Shi H, et al. Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci, 2018,75(19):3593-3607. doi: 10.1007/s00018-018-2810-3 pmid: 29637228

[8]	Xu R, Qin R, Li H, et al. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J, 2017,15(6):713-717. doi: 10.1111/pbi.12669 pmid: 27875019

[9]	Gao L, Cox D B T, Yan W X, et al. Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol, 2017,35(8):789-792. doi: 10.1038/nbt.3900 pmid: 28581492

[10]	Yamano T, Zetsche B, Ishitani R, et al. Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1. Moleculer Cell, 2017,67(4):633-645.

[11]	Lei C, Li S Y, Liu J K, et al. The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted ligation) method for efficient editing of large DNA constructs in vitro. Nucleic Acids Res, 2017,45(9):e74. doi: 10.1093/nar/gkx018 pmid: 28115632

[12]	Moreno-Mateos M A, Fernandez J P, Rouet R, et al. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun, 2017,8(1):2024. doi: 10.1038/s41467-017-01836-2 pmid: 29222508

[13]	Kim D, Kim J, Hur J K, et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol, 2016,34(8):863-868. doi: 10.1038/nbt.3609 pmid: 27272384

[14]	Kleinstiver B P, Tsai S Q, Prew M S, et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol, 2016,34(8):869-874. doi: 10.1038/nbt.3620 pmid: 27347757

[15]	Dong D, Ren K, Qiu X, et al. The crystal structure of Cpf1 in complex with CRISPR RNA. Nature, 2016,532(7600):522-526. doi: 10.1038/nature17944 pmid: 27096363

[16]	Yamano T, Nishimasu H, Zetsche B, et al. Crystal Structure of Cpf1 in complex with Guide RNA and Target DNA. Cell, 2016,165(4):949-962. doi: 10.1016/j.cell.2016.04.003 pmid: 27114038

[17]	Singh D, Mallon J, Poddar A, et al. Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc Natl Acad Sci U S A, 2018,115(21):5444-5449. doi: 10.1073/pnas.1718686115 pmid: 29735714

[18]	Stella S, Alcón P, Montoya G. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage. Nature, 2017,547(7664):476. doi: 10.1038/nature23300 pmid: 28678773

[19]	Hu X, Wang C, Liu Q, et al. Targeted mutagenesis in rice using CRISPR-Cpf1 system. J Genet Genomics, 2017,44(1):71-73. doi: 10.1016/j.jgg.2016.12.001 pmid: 28043782

[20]	Kim Y, Cheong S A, Lee J G, et al. Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol, 2016,34(8):808-810. doi: 10.1038/nbt.3614 pmid: 27272387

[21]	Watkins-Chow D E, Varshney G K, Garrett L J, et al. Highly efficient Cpf1-mediated gene targeting in mice following high concentration pronuclear injection. G3 (Bethesda), 2017,7(2):719-722. doi: 10.1534/g3.116.038091

[22]	Hur JK, Kim K, Been K W, et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol, 2016,34(8):807-808. doi: 10.1038/nbt.3596 pmid: 27272385

[23]	Liu C, Zhang L, Liu H, et al. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.[J]. Controlled Release, 2017,266(11):17-26. doi: 10.1016/j.jconrel.2017.09.012

[24]	Safari F, Zare K, Negahdaripour M, et al. CRISPR Cpf1 proteins: structure, function and implications for genome editing. Cell Biosci, 2019,9:36. doi: 10.1186/s13578-019-0298-7 pmid: 31086658

[25]	Li T, Zhu L, Xiao B, et al. CRISPR-Cpf1-mediated genome editing and gene regulation in human cells. Biotechnol Adv. 2019,37(1):21-27. doi: 10.1016/j.biotechadv.2018.10.013 pmid: 30399413

[26]	Gao Z, Herrera-Carrillo E, Berkhout B. Improvement of the CRISPR-Cpf1 system with ribozyme-processed crRNA RNA Biol 2018,15(12):1458-1467. doi: 10.1080/15476286.2018.1551703 pmid: 30470168

[27]	Fernandez J P, Vejnar C E, Giraldez A J et al. Optimized CRISPR-Cpf1 system for genome editing in zebrafish. Methods. 2018,150(11):11-18. doi: 10.1016/j.ymeth.2018.06.014

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