Author :
Abstract
The development of CRISPR/Cas9 system has revolutionized and accelerated the era of programmable nuclease mediated gene editing technologies. The CRISPR/Cas9 system consists of a Cas9 nuclease and a synthetic chimeric RNA known as guide RNA (gRNA). Cas9 endonuclease cleaves target DNA at a sequence-specific site with the help of designed sgRNA. The utilization of Cas9 nuclease protein as an important molecular tool in gene editing makes a great emphasis on the production of recombinant Cas9 protein. Due to Cas9 protein’s large size and its complexity, recombinant Cas9 protein production can be challenged. Here, we subcloned a sequence encoding human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9), and recombinant protein production of hSpCas9 was evaluated in different E. coli strains at different induction settings. The expression of recombinant hSpCas9 protein was determined within BL21(DE3) host strain at 25 °C induction temperature for four hours and following overnight induction. The purification of HisTagged recombinant hSpCas9 protein was performed by affinity chromatography. These findings imply that recombinant hSpCas9 protein could be produced on a large scale for potential utilization in the biotechnology market. The production of recombinant hSpCas9 nuclease protein could provide a significant tool for the advancement in the ribonucleoprotein complex utilization in CRISPR/Cas9 technology as well as the improvement of pharmaceutical-based approaches to modulate Cas9 nuclease protein activity for precise gene editing.
Keywords
Abstract
The development of CRISPR/Cas9 system has revolutionized and accelerated the era of programmable nuclease mediated gene editing technologies. The CRISPR/Cas9 system consists of a Cas9 nuclease and a synthetic chimeric RNA known as guide RNA (gRNA). Cas9 endonuclease cleaves target DNA at a sequence-specific site with the help of designed sgRNA. The utilization of Cas9 nuclease protein as an important molecular tool in gene editing makes a great emphasis on the production of recombinant Cas9 protein. Due to Cas9 protein’s large size and its complexity, recombinant Cas9 protein production can be challenged. Here, we subcloned a sequence encoding human codon-optimized Streptococcus pyogenes Cas9 (hSpCas9), and recombinant protein production of hSpCas9 was evaluated in different E. coli strains at different induction settings. The expression of recombinant hSpCas9 protein was determined within BL21(DE3) host strain at 25 °C induction temperature for four hours and following overnight induction. The purification of HisTagged recombinant hSpCas9 protein was performed by affinity chromatography. These findings imply that recombinant hSpCas9 protein could be produced on a large scale for potential utilization in the biotechnology market. The production of recombinant hSpCas9 nuclease protein could provide a significant tool for the advancement in the ribonucleoprotein complex utilization in CRISPR/Cas9 technology as well as the improvement of pharmaceutical-based approaches to modulate Cas9 nuclease protein activity for precise gene editing.
Keywords
- Carmignotto, G. P., & Azzoni, A. R. (2019). On the expression of
- Carmignotto, G. P., & Azzoni, A. R. (2019). On the expression ofrecombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3)Rosetta strains. Journal of Biotechnology, 306, 62–70. https://doi.org/https://doi.org/10.1016/j.jbiotec.2019.09.012
- Chira, S., Gulei, D., Hajitou, A., & Berindan-Neagoe, I. (2018).Restoring the p53 ‘Guardian’ Phenotype in p53-Deficient TumorCells with CRISPR/Cas9. Trends in Biotechnology, 36(7), 653– 660. https://doi.org/10.1016/j.tibtech.2018.01.014
- Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targetedgenome engineering in human cells with the Cas9 RNA-guidedendonuclease. Nature Biotechnology, 31(3), 230–232. https://doi.org/10.1038/nbt.2507
- Chu, V. T., Weber, T., Graf, R., Sommermann, T., Petsch, K.,Sack, U., Volchkov, P., Rajewsky, K., & Kühn, R. (2016). Efficientgeneration of Rosa26 knock-in mice using CRISPR/Cas9 inC57BL/6 zygotes. BMC Biotechnology, 16(1), 4. https://doi.org/10.1186/s12896-016-0234-4
- Cyranoski, D. (2016). CRISPR gene-editing tested in a person forthe first time. Nature, 539(7630), 479. https://doi.org/10.1038/nature.2016.20988
- DeWitt, M. A., Corn, J. E., & Carroll, D. (2017). Genome editingvia delivery of Cas9 ribonucleoprotein. Methods, 121–122, 9–15. https://doi.org/https://doi.org/10.1016/j.ymeth.2017.04.003
- Glass, Z., Lee, M., Li, Y., & Xu, Q. (2018). Engineering theDelivery System for CRISPR-Based Genome Editing. Trends inBiotechnology, 36(2), 173–185. https://doi.org/10.1016/j.tibtech.2017.11.006
- Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., &Charpentier, E. (2012). A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity. Science (NewYork, N.Y.), 337(6096), 816–821. https://doi.org/10.1126/science.1225829
- Jinek, M., Jiang, F., Taylor, D. W., Sternberg, S. H., Kaya, E., Ma,E., Anders, C., Hauer, M., Zhou, K., Lin, S., Kaplan, M., Iavarone,A. T., Charpentier, E., Nogales, E., & Doudna, J. A. (2014).Structures of Cas9 endonucleases reveal RNA-mediatedconformational activation. Science (New York, N.Y.), 343(6176), 1247997. https://doi.org/10.1126/science.1247997
- Kane, J. F. (1995). Effects of rare codon clusters on high-levelexpression of heterologous proteins in Escherichia coli. CurrentOpinion in Biotechnology, 6(5), 494–500.https://doi.org/https://doi.org/10.1016/0958-1669(95)80082-4
- Karakus, C., Uslu, M., Yazici, D., & Salih, B. A. (2016).Evaluation of immobilized metal affinity chromatography kitsfor the purification of histidine-tagged recombinant CagAprotein. Journal of Chromatography B, 1021, 182–187.https://doi.org/https://doi.org/10.1016/j.jchromb.2015.11.045
- Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014). Highlyefficient RNA-guided genome editing in human cells via deliveryof purified Cas9 ribonucleoproteins. Genome Research, 24(6), 1012–1019. https://doi.org/10.1101/gr.171322.113
- Kim, S. M., Shin, S. C., Kim, E. E., Kim, S.-H., Park, K., Oh, S. J.,& Jang, M. (2018). Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for CancerTreatment. ACS Nano, 12(8), 7750–7760. https://doi.org/10.1021/acsnano.8b01670
- Li, L., Hu, S., & Chen, X. (2018). Non-viral delivery systems forCRISPR/Cas9-based genome editing: Challenges andopportunities. Biomaterials, 171, 207–218. https://doi.org/10.1016/j.biomaterials.2018.04.031
- Liang, X., Potter, J., Kumar, S., Zou, Y., Quintanilla, R.,Sridharan, M., Carte, J., Chen, W., Roark, N., Ranganathan, S.,Ravinder, N., & Chesnut, J. D. (2015). Rapid and highly efficientmammalian cell engineering via Cas9 protein transfection.Journal of Biotechnology, 208, 44–53. https://doi.org/https://doi.org/10.1016/j.jbiotec.2015.04.024
- Lin, S., Staahl, B. T., Alla, R. K., & Doudna, J. A. (2014).Enhanced homology-directed human genome engineering bycontrolled timing of CRISPR/Cas9 delivery. ELife, 3, e04766. https://doi.org/10.7554/eLife.04766
- Liu, C., Zhang, L., Liu, H., & Cheng, K. (2017). Deliverystrategies of the CRISPR-Cas9 gene-editing system fortherapeutic applications. Journal of Controlled Release, 266, 17–26. https://doi.org/https://doi.org/10.1016/j.jconrel.2017.09.012
- Ma, E., Harrington, L. B., O’Connell, M. R., Zhou, K., & Doudna,J. A. (2015). Single-Stranded DNA Cleavage by DivergentCRISPR-Cas9 Enzymes. Molecular Cell, 60(3), 398–407. https://doi.org/10.1016/j.molcel.2015.10.030
- Maddalo, D., Manchado, E., Concepcion, C. P., Bonetti, C.,Vidigal, J. A., Han, Y.-C., Ogrodowski, P., Crippa, A., Rekhtman,N., de Stanchina, E., Lowe, S. W., & Ventura, A. (2014). In vivoengineering of oncogenic chromosomal rearrangements with theCRISPR/Cas9 system. Nature, 516(7531),423–427. https://doi.org/10.1038/nature13902
- Mali, P., Esvelt, K. M., & Church, G. M. (2013).
- Pawluk, A., Amrani, N., Zhang, Y., Garcia, B., Hidalgo-Reyes, Y.,Lee, J., Edraki, A., Shah, M., Sontheimer, E. J., Maxwell, K. L., &Davidson, A. R. (2016). Naturally Occurring Off-Switches forCRISPR-Cas9. Cell, 167(7), 1829-1838.e9. https://doi.org/10.1016/j.cell.2016.11.017
- Philippe, H., & Rodolphe, B. (2010). CRISPR/Cas, the ImmuneSystem of Bacteria and Archaea. Science, 327(5962), 167–170. https://doi.org/10.1126/science.1179555
- Prashant, M., Luhan, Y., M., E. K., John, A., Marc, G., E., D. J., E.,N. J., & M., C. G. (2013). RNA-Guided Human GenomeEngineering via Cas9. Science, 339(6121), 823–826.Ramakrishna, S., Kwaku Dad, A.-B., Beloor, J., Gopalappa, R.,Lee, S.-K., & Kim, H. (2014). Gene disruption by cell-penetratingpeptide-mediated delivery of Cas9 protein and guide RNA.Genome Research, 24(6), 1020–1027. https://doi.org/10.1101/gr.171264.113
- Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S.,Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S.,Koonin, E. V, Sharp, P. A., & Zhang, F. (2015). In vivo genome
- Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems forediting, regulating and targeting genomes. Nature Biotechnology, 32(4), 347–355. https://doi.org/10.1038/nbt.2842
- Staff, T. P. O. N. E. (2014). Correction: Efficient Mutagenesis byCas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs. PLOS ONE, 9(8), e106396. https://doi.org/10.1371/journal.pone.0106396
- Tegel, H., Tourle, S., Ottosson, J., & Persson, A. (2010). Increasedlevels of recombinant human proteins with the Escherichia colistrain Rosetta(DE3). Protein Expression and Purification, 69(2), 159–167.
- Tripathi, N. K. (2016). Production and Purification ofRecombinant Proteins from Escherichia coli. ChemBioEngReviews, 3(3), 116–133. https://doi.org/https://doi.org/10.1002/cben.201600002
- Uslu, M., Siyah, P., Harvey, A. J., & Kocabaş, F. (2021). ChapterSix - Modulating Cas9 activity for precision gene editing. In V. B.T.-P. in M. B. and T. S. Singh (Ed.), Reprogramming theGenome: CRISPR-Cas-based Human Disease Therapy (Vol. 181,pp. 89–127). Academic Press.https://doi.org/https://doi.org/10.1016/bs.pmbts.2021.01.015
- Yang, S., Chang, R., Yang, H., Zhao, T., Hong, Y., Kong, H. E.,Sun, X., Qin, Z., Jin, P., Li, S., & Li, X.-J. (2017). CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse modelof Huntington’s disease. The Journal of Clinical Investigation, 127(7), 2719–2724. https://doi.org/10.1172/JCI92087
- and Clapp 2018; Solanki, et al. 2017). The incidence of FA range from 1 in 100000 to 250000 births (Anurogo, et al. 2021).
