In-Vitro Validation of CRISPR-Mediated Cleavage of Integrin ITGβ3
Department
Biological Sciences
Advisor
Paula Soneral
Document Type
Poster
Version
Metadata Only
Abstract
Breast cancer is growing worldwide, requiring new avenues of treatment. During metastasis, breast cancer cells use the integrin family of receptors for adhesion to the extracellular matrix. Prior RNA-seq experiments and differential gene expression analysis of the MCF-7 breast cancer cell line identified the ITGβ3 integrin gene as overexpressed. This research study aims to determine whether ITGβ3 plays a functional role in cell adhesion and migration. We hypothesize that knockdown of ITGβ3 causes MCF-7 breast cancer cells to deadhere from substrata. To test this hypothesis, prior work designed four CRISPR guide RNAs (gRNAs) for ITGβ3, two targeting the promoter (54/fw and 64/fw), and two targeting exon 2 (41/rev and 35/rev). To validate the target ITGβ3 DNA cleavage, we used in-vitro CRISPR-Cas9 nuclease assays containing synthesized gRNA, Cas9 enzyme, and a PCR-amplified ITGβ3 target sequence. After validating cleavage activity in vitro, we transfected MCF-7 breast cancer cells with the CRISPR-Cas9 complex. Gel electrophoresis of our in-vitro nuclease assay revealed the 41/rev CRISPR had the strongest cleavage, and 54/fw and 64/fw and 35/rev gRNAs showed partial cleavage. Cells transfected with a 1:1 Cas9:gRNA and 2:1 Cas9:gRNA ratio of 41/rev showed some evidence of disruption to cell adhesion among cells that were 65% and 85% viable respectively. These preliminary data, if reproducible, suggest that ITGβ3 may play a role in cell adhesion and serve as a valuable biomarker for breast cancer metastasis.
Recommended Citation
Simmonds, Harrison and Soneral, Paula, "In-Vitro Validation of CRISPR-Mediated Cleavage of Integrin ITGβ3" (2024). Science Symposium. 34.
https://spark.bethel.edu/science_symposium/spring2024/schedule2024/34
Terms of Use and License Information
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
In-Vitro Validation of CRISPR-Mediated Cleavage of Integrin ITGβ3
Breast cancer is growing worldwide, requiring new avenues of treatment. During metastasis, breast cancer cells use the integrin family of receptors for adhesion to the extracellular matrix. Prior RNA-seq experiments and differential gene expression analysis of the MCF-7 breast cancer cell line identified the ITGβ3 integrin gene as overexpressed. This research study aims to determine whether ITGβ3 plays a functional role in cell adhesion and migration. We hypothesize that knockdown of ITGβ3 causes MCF-7 breast cancer cells to deadhere from substrata. To test this hypothesis, prior work designed four CRISPR guide RNAs (gRNAs) for ITGβ3, two targeting the promoter (54/fw and 64/fw), and two targeting exon 2 (41/rev and 35/rev). To validate the target ITGβ3 DNA cleavage, we used in-vitro CRISPR-Cas9 nuclease assays containing synthesized gRNA, Cas9 enzyme, and a PCR-amplified ITGβ3 target sequence. After validating cleavage activity in vitro, we transfected MCF-7 breast cancer cells with the CRISPR-Cas9 complex. Gel electrophoresis of our in-vitro nuclease assay revealed the 41/rev CRISPR had the strongest cleavage, and 54/fw and 64/fw and 35/rev gRNAs showed partial cleavage. Cells transfected with a 1:1 Cas9:gRNA and 2:1 Cas9:gRNA ratio of 41/rev showed some evidence of disruption to cell adhesion among cells that were 65% and 85% viable respectively. These preliminary data, if reproducible, suggest that ITGβ3 may play a role in cell adhesion and serve as a valuable biomarker for breast cancer metastasis.