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USE OF CRISPR / CAS9 (CLUSTERS OF REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS) FOR GENOMIC EDITION OF Physcomitrella patens FOR LONG-SCALE EXPRESSION OF ANTIMICROBIAL PEPTIDES
Catholic University of Brasilia- UCB
Biotechnologies and Informatics
Genomic Science and Biotechnology
Simoni Campos Dias and Nicolau Brito da Cunha
Simoni Campos Dias and Nicolau Brito da Cunha
Type of Research Project
- Basic science
What is the background of the project?
Agriculture is an essential sector of the Brazilian economy, contributing to economic growth and exchange rate equilibrium and a significant part has been concentrated in the livestock sector, mainly in the production of beef, pork and poultry. However, Brazilian production has been threatened by several pathogens and pests that cause losses year after year in both of them. Therefore, the development of new tools to control these pathogens is important, and with the use of biotechnology one can find molecules that have antimicrobial activity. However, one of the major obstacles to the production of large-scale antimicrobial peptides is finding an cost-effective production system. The production of proteins using the moss Physcomitrella patens, a bryophyte model, has good cost-benefit, mainly by allowing the use of bioreactors. In this way, the objective of this project is to establish the stable transformation conditions of the P. patens moss using the CRISPR-Cas9 system and to express molecules with antimicrobial action as proof of concept. Finally, this work will contribute to the improvement of biotechnological systems for the development and production of anti-infectives, as it will take into account cutting-edge technologies, aiming at the production of safe and quality food.
What is the aim of the project?
Establish the genetic transformation of moss Physcomitrella patens using the CRISPR / Cas9 (clusters of regularly interspaced short palindromic repeats) genome-editing system for heterologous expression on a large scale of antimicrobial peptides, chosen from a peptide bank generated from the Pantanal and Cerrado biome. The creation and validation of this bank is one of the objectives of the INCT-Bioinspir (National Institute of Science and Technology) project.
What techniques and methods are used?
Firstly, to test the primary hypothesis, amplification of DNA fragments containing the antimicrobial peptide coding sequences will be performed. This step is essential for obtaining satisfactory amounts of DNA for gene cloning. The fragments will then be subcloned into adaptive vectors (plasmids) to enable further manipulation in vitro. This step will be conducted by ligating the amplicons to the plasmids typically designed to receive PCR amplified DNA fragments, followed by genetic transformation of competent Escherichia cells and selection in culture medium with correct antibiotics. The main objective of this step is to obtain micrograms of recombinant DNA for further manipulations. The inserts will then be released from the recombinant plasmids by enzymatic digestion of the DNA with restriction endonucleases. This must be done to obtain DNA fragments of interest with compatible ends for insertion into plasmids containing the genetic elements suitable for high levels of gene expression in moss. The next step will be the binding of the plasmid-derived inserts to the gene expression in moss, followed by genetic transformation of competent bacterial cells and gene cloning into selection media, with subsequent inspection of the presence of the insert by PCR, digestion with restriction endonucleases and DNA sequencing. All to ensure that the gene construct presents itself with the correct structure and sequence. To test the second hypothesis will genetically transform P. patens using driselase enzymes to digests the cell wall of P. patens cells, as indicated below. After obtaining satisfactory amounts of DNA (micrograms) will be performed the genetic transformation of P. patens with plasmids containing the whole system of gene expression and genome editing by CRISPRs. It is expected to obtain transformants events from selected moss in culture media that have their genome edited by the CRISPR technique, in which the genes encoding antimicrobial peptides will undergo site recombination in genomic hot spot for high gene expression. Thirdly, to check the levels of gene expression at the transcriptional level, the Real Time PCR technique will be performed. This technique allows the evaluation of the biosynthesis rate of mRNA copies corresponding to the primary transcripts of the gene of interest, and is fundamental to evaluate if the gene expression in moss presents in its first level. To perform the RT-PCR, we will extract total RNA, construct specific cDNA and evaluate gene expression by Reverse Transcription Polymerase Chain Reaction (RT-PCR). It will then be necessary to check for transgene expression at the translational level and characterize the peptide structures for their masses and amino acid sequences. This will be evaluated using a mass spectrometer called by MALDI-TOF TOF which mechanism will work by measuring an ion’s mass-to-charge, as well as, providing peptide sequence information, and also purify the antimicrobial peptides using High performance liquid chromatography (HPLC).
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student should be able to follow graduate students in a broad discussion about the project. It is also expected that they can be able to run some bench experiments, such as knowing how to prepare different gels, execute RT-PCR procedure, interpret MALDI TOF TOF, mass spectrometer results, and other procedures involved in establishing the conditions for transformation of P. patens protoplasts using CRISPR / Cas9 and expressing antimicrobial peptides from P. patens which was transformed via CRISPR / Cas9. The experiments will always be supervised by graduate students. The outcome of the student participation should be a seminar presented to our research group. The seminar must cover the topic of the project.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
It is planned some discussions based on papers shared with students during the exchange program. The discussion will be lead by the project coordinator. Students will be also invited to present a seminar to other students about their course and the reasons he/she decided to come to our group in the end of the exchange program.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
We are not expecting any special skill more than that a medical student must have. There is no legal limitation we can envisage.But,it will be useful for the student to have knowledge of searching on databases to improve his knowledge about the aim of the project. Also, to know at least theoretically, some basic laboratory procedures such as RT-PCR, how a mass spectrometer works it’s required for an student to know.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Students in biomedical fields
- Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens. Collonnier C; Guyon-Debast A; Maclot F; Mara K; Charlot F; Nogué F. Methods. 2017 May 15;121-122:103-117. doi: 10.1016/j.ymeth.2017.04.024. Epub 2017 May 4.
- Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens. Lopez-Obando M; Hoffmann B; Géry C; Guyon-Debast A; Téoulé E; Rameau C; Bonhomme S; Nogué F. G3 (Bethesda). 2016 Nov 8;6(11):3647-3653. doi: 10.1534/g3.116.033266.
- CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens. Collonnier C; Epert A; Mara K; Maclot F; Guyon-Debast A; Charlot F; White C; Schaefer DG; Nogué F. Plant Biotechnol J. 2017 Jan;15(1):122-131. doi: 10.1111/pbi.12596. Epub 2016 Jul 22. vesicles. Nature reviews. Immunology 14:195-208
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