Generation of gene-deficient mice by using CRISPR/Cas genome editing

• Grant number: 15/04596-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: June 08, 2015
• End date: October 07, 2015
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Dario Simões Zamboni
• Grantee: Catarina Veltrini Horta
• Supervisor: Hidde Ploegh
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Whitehead Institute for Biomedical Research, United States
• Associated to the scholarship: 12/02786-9 - The participation of innate immune receptors in the autophagy induction in response to Trypanosoma cruzi infection., BP.DR

Abstract

Autophagy is a process of cellular degradation that captures and degrades organelles and intracellular pathogens and delivers them to autolysosomes for destruction. The role of autophagy in Trypanosoma cruzi infection is still largely unknown. During the development of the Ph.D. project, we found that autophagy is induced in response to T. cruzi favoring restriction of parasite replication by macrophages. Moreover, we demonstrated that multiple pattern recognition receptors (PRRs) as well as nitric oxide (NO) production are required for autophagy induction by T. cruzi. In this context, we intend to determinate the mechanisms involved in the NO-mediated autophagy induction. For this proposes, we are carrying mass spectrometry analysis to identify novel proteins that can be specifically regulated by NO in response to T. cruzi infection. The next and important step in this project is to validate the candidates by generating gene deficient-mice. The fast and easy way to do this involves the use of CRISPR/Cas (clustered regularly interspaced repeats - Cas associated) genome editing technology. To the best of our knowledge, CRISPR-mediated genome editing is still not available in Brazil, although over the last 5 years CRISPR/Cas has been extensively used to perform genome engineering in eukaryotic cells and organism in short time, but only recently it has been used to generate a gene-modified mouse. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 to virtually anywhere the genome. Upon targeting, Cas9 generates DNA double-strand breaks (DSBs) and facilitates the repair or insertion of mutations. In this project, we aim the training the recipient of this Ph.D. fellowship for performing CRISPR/Cas technology to address key questions related to the mechanisms involved in the NO/autophagy mediated restriction of parasite replication. The development of this project may ultimately facilitate a better comprehension of host immune responses mediated by NO and autophagy and the training of an individual that can further establish this technique in Brazil, thus favoring the scientific development of the scientific community in São Paulo estate. Of note, we decided to acquire the CRISPR/Cas technology in the Hidde Ploegh's laboratory, which work in close collaboration with Rudolf Jaeninisch's laboratory at Whitehead Institute, CA, Massachusetts, the pioneers in generate gene-targeted mice by CRISPR. These labs are fully proficient at performing both somatic cell nuclear transfer and CRISPR/Cas genome editing in mouse zygotes and showed a great enthusiasms to collaborate with our group to help the establishment of this technique in Brazil. (AU)