Period
2019 - 2022
Colaborators
Responsible: Raquel Pinho
PI: Dr.Elizabeth Maga, University of Californa, Davis, CA - United States
PI: Dr. Pablo Ross, University of Californa, Davis, CA - United States
PI: Dr. Trish Berger, University of Californa, Davis, CA - United States
PI: Dr. James Murray, University of Californa, Davis, CA - United States
Colaborator: Dr. Cicera Lazzarotto, St.Jude Children’s Research Hospital, Mephis, TN - United States
Colaborator: Dr. Shengdar Tsai, St.Jude Children’s Research Hospital, Mephis, TN - United States
Colaborator: Barbara Jean-Nitta, University of Californa, Davis, CA - United States
Colaborator: Dr. Insung Park, University of Californa, Davis, CA - United States
Colaborator: Dr. Kelly Zacanti, University of Californa, Davis, CA - United States
Colaborator: Barbara Jean-Nitta, University of Californa, Davis, CA - United States
Colaborator: Jennifer Jankovitz , University of Californa, Davis, CA - United States
Location
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University of California, Davis, CA - United States
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St.Jude Children’s Research Hospital, Mephis, TN - United States
Raquel Pinho’s main responsabilities
- Developing methodology for detection of off-target events of CRISPR/Cas9 system in porcine model.
- Pig oocyte collection, maturation and activation for development into parthenotes and posterior electroporation of CRISPR/Cas9 components.
- PCR of possible off-target sites for Cas9 in vitro assay experiments
- Genotyping of pig embryos and fetuses at on-target and off-target sites by sanger sequencing and PacBio SMRT Sequencing.
- Assay development for guideRNA/Cas9 efficiency using qPCR and ddPCR.
- Development of multiplex rhAmpSeq PCR panel for analysis off-target sites.
- Management of molecular biology laboratory.
Description
In this project, we used the CRISPR/Cas9 system targeting a porcine gene in zygotes for the production of a AR knock-out pig. Two different gRNAs were targeting the exon 1 of the gene, and cleavage efficiency, mutation frequency and off-target effects were analyzed, first with in vitro and in vivo approaches using blastocysts and parthenotes. Embryos and fetuses prevenient from failed pregnancies were also analyzed for the presence of mutation in the target site and probable off targets positions. Sanger sequencing was used to confirm reference sequences for target and off-target sites and mutations. CHANGE-seq was done in three wild-type pigs (two females and one male) for the genome wide analysis of off-target sites. The sites observed in the CHANGE-seq protocol will be sequenced in blastocysts, embryos and fetuses injected with the guides used in the study, using amplicon deep sequencing (rhAmpSeq).
Data
Data not published yet
Publications
Data not published yet