sgRNA design and in vitro nucleolytic analysis of the Cas9‐RNP complex for transgene‐free genome editing of the eIF4E1 gene from Capsicum an‐ nuum L.

https://doi.org/10.22146/ijbiotech.86778

Josefanny Tham(1), Alfred Patisenah(2), Tommy Octavianus Soetrisno Tjia(3), Santiago Signorelli(4), Intan Taufik(5), Karlia Meitha(6*)

(1) School of Life Sciences and Technology, Institut Teknologi Bandung, West Java 40132, Indonesia
(2) School of Life Sciences and Technology, Institut Teknologi Bandung, West Java 40132, Indonesia
(3) School of Life Science and Technology, Tokyo Institute of Technology, Japan 152-8550
(4) Departamento de Biología Vegetal, Universidad de la República, Uruguay 11200
(5) School of Life Sciences and Technology, Institut Teknologi Bandung, West Java 40132, Indonesia
(6) School of Life Sciences and Technology, Institut Teknologi Bandung, West Java 40132, Indonesia
(*) Corresponding Author

Abstract


Chili (Capsicum annuum L.) is a highly valued vegetable, renowned for its unique taste and aroma. However, chili production faces challenges in meeting the high demand due to infections caused by pathogens such as ChiVMV (potyvirus). Previous studies have suggested that chili eIF4E1 plays a crucial role in potyvirus gene transcription. Therefore, this study explores the potential of CRISPR‐Cas9‐based genome editing to enhance chili resistance by introducing premature stop codons or truncated proteins. Two sgRNAs were designed, targeting the first and second intron of the eIF4E1 gene. The production of Cas9 protein was assessed with varying IPTG concentrations in Escherichia coli BL21(DE3), carrying 4xNLS‐pMJ915v2‐sfGFP plasmid with a TEV protease cut‐site at the N terminal. The findings indicate that the optimal IPTG concentration is 500 µM. Purification using an IMAC column confirmed the presence of Cas9 in the initial 2 mL of the eluted fractions, as indicated by numerous background proteins. Nevertheless, successful formation of Cas9‐RNP complexes was achieved for both sgRNAs. The nucleolytic activity of Tag‐Cas9 (carrying the MBP‐tag) and Cas9 was confirmed through in vitro endonuclease activity assays. The next step involved transfecting chili protoplasts with these RNP complexes to edit the chili eIF4E1 gene.


Keywords


CRISPR‐Cas9; Endonuclease; Recombinant protein; sgRNA Cas9‐RNP



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Cao H, Lin R. 2009. Quantitative evaluation of histag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells. Biotechnol. Prog. 25(2):461–467.

doi:10.1002/btpr.121.

Carmignotto GP, Azzoni AR. 2019. On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains. J. Biotechnol. 306:62– 70. doi:10.1016/j.jbiotec.2019.09.012.

Cribbs AP, Perera SM. 2017. Science and bioethics of CRISPR-CAS9 gene editing: An analysis towards separating facts and fiction. Yale J. Biol. Med. 90(4):625–634.

da Costa DV, Paiva CLdA, Bento CdS, Sudré CP, Cavalcanti TFM, Gonçalves LSA, Viana AP, Rodrigues R. 2021. Breeding for pepper yellow mosaic virus resistance and agronomic attributes in recombinant inbred lines of chili pepper (Capsicum baccatum L.) using mixed models. Sci. Hortic. (Amsterdam). 282:110025. doi:10.1016/j.scienta.2021.110025.

Dinh T, Bernhardt TG. 2011. Using superfoldergreen fluorescent protein for periplasmic protein localization studies. J. Bacteriol. 193(18):4984–7.doi:10.1128/JB.00315-11.Donovan RS, Robinson CW, Click BR. 1996. Review: Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J. Ind. Microbiol. 16(3):145–54. doi:10.1007/BF01569997.

Duprat A, Caranta C, Revers F, Menand B, Browning KS, Robaglia C. 2002. The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. Plant J. 32(6):927–34. doi:10.1046/j.1365- 313X.2002.01481.x.

Flottmann F, Pohl GM, Gummert J, Milting H, Brodehl A. 2022. A detailed protocol for expression, purification, and activity determination of recombinant SaCas9. STAR Protoc. 3(2):101276. doi:10.1016/j.xpro.2022.101276.

Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. 2013. High-frequency offtarget mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31:822–826. doi:10.1038/nbt.2623.

Groves NR, Mckenna JF, Evans DE, Graumann K, Meier I. 2019. A nuclear localization signal targets tailanchored membrane proteins to the inner nuclear envelope in plants. J. Cell Sci. 132(7):jcs226134. doi:10.1242/jcs.226134.

Hayat SMG, Farahani N, Golichenari B, Sahebkar

A. 2018. Recombinant protein expressionin Escherichia coli (E.coli): What we needto know. Curr. Pharm. Des. 24(6):718–725.doi:10.2174/1381612824666180131121940.Hsu PD, Lander ES, Zhang F. 2014. Development and applications of CRISPR-Cas9 forgenome engineering. Cell 157(6):1262–1278.doi:10.1016/j.cell.2014.05.010.Jeong H, Kim HJ, Lee SJ. 2015. Completegenome sequence of Escherichia coli strainBL21. Genome Announc. 3(2):e00134–15.



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