D3-a - Genomic, Proteomic, and Functional Validation of CRISPR/Cas9 Knockouts in Mammalian Cell Lines

Saturday, October 11, 2025

10:00 AM - 12:00 PM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Titan Yasir

High School Researcher
Rice University
Houston, Texas, United States

Co-Author(s)

Melissa S. CantÃº

Graduate Student
Rice University
Houston, Texas, United States
Abigail Fabiano

Graduate Student
Rice University, United States

Last Author(s)

Michael King

Professor
Rice University, United States

Introduction: : Piezo1:
Mechanical forces, such as shear stress, within the bloodstream play a critical role in regulating immune cell behavior [1]. For instance, Piezo1 is a mechanosensitive ion channel that changes conformation in response to mechanical force, allowing calcium influx [1]. Piezo1 activation amplifies the T cell receptor signal transduction cascade and, consequently, enhances T cell activation, proliferation, and cytotoxicity [2]. To investigate how Piezo1 influences T cell function under physiological fluid shear stress (FSS), a key step is to generate a stable PIEZO1 knockout (KO) Jurkat cell line to enable controlled functional studies.

Calretinin:
Metastasis—the spread of cancer to distant organs [3]—is partly enabled by the ability of tumor cells to survive FSS of the bloodstream [4]. In some cancers, such as pancreatic cancer, the role of calretinin in promoting metastasis is a growing area of research; Calretinin is a calcium-binding protein encoded by the gene CALB2 upregulated by FSS [5]. To elucidate the mechanistic contribution of calretinin in prostate cancer cells upon FSS stimulation, CALB2 was knocked out in PC3 cells, cancer cells derived from prostate cancer bone metastases [6], to assess its potential protective role against FSS in the bloodstream.

Materials and

Methods: : CRISPR/Cas9 KOs for PIEZO1 and CALB2 were produced using the lentiCRISPRv2 system [7], [8]. Following Cas9-induced double-strand breaks, the cell's primary repair pathway—non-homologous end joining (NHEJ)—frequently introduces small insertions or deletions (indels) at the cut site. These indels often result in frameshift mutations in the reading frame, producing a loss-of-function/KO [9]. sgRNAs targeting each gene were cloned into these lentiviral vectors, which were then used to produce lentiviral particles for transducing Jurkat and PC3 cells. Engineered cells were purified via fluorescence-activated cell sorting (FACS).

For both cell types, genomic DNA was extracted, target exons were PCR amplified, and Sanger sequenced, with editing efficiency quantified using ICE software (PC3) or TIDE software (Jurkat).

PC3 cell lysate was used for Western Blot analysis using a primary antibody against calretinin. Western Blot membranes were imaged using the ChemiDoc Imaging System (BioRad), and protein quantification was carried out using the Fiji software, normalizing to GAPDH.

Functional validation of Piezo1 was performed by loading Jurkat cells with the calcium-responsive dye Fluo-4, which fluoresces green upon binding intracellular calcium. Live cell fluorescence imaging was conducted before and after stimulation with Yoda1, a Piezo1-specific agonist that activates Piezo1 and thus induces calcium influx. Calcium responses in KO cells were compared to those in wild type controls to assess Piezo1 functional disruption.

All experiments included replicates and appropriate controls to ensure reproducibility.

Results, Conclusions, and Discussions:: Calretinin:
Shown in Figure 1A, genomic validation using ICE analysis showed high indel efficiency of 91% in both FSS and non-FSS PC3 cells, confirming successful CALB2 knockout across all cell populations tested. The R² values of 0.95 (non-FSS) and 0.91 (FSS) indicated excellent model fit. Western blot analysis shown in Figures 1B and 1C corroborate successful CALB2 KO, with lower calretinin expression in KO groups compared to WT counterparts. Furthermore, quantification of Calretinin:GAPDH ratios confirmed that FSS greatly elevates calretinin in WT cells, but this mechanically induced increase is significantly reduced in CALB2 KO cells, indicating that CALB2 is necessary for full mechanotransduction response at the protein level.

Piezo1:
As shown in Figure 1D, TIDE analysis revealed 2.5% editing efficiency at the PIEZO1 locus with 96.3% of sequences unchanged, suggesting low CRISPR cutting efficiency potentially due to effective NHEJ repair. The R² value of 0.99 indicated excellent model fit with statistically significant changes (p < 0.001). However, as shown in Figure 1E, functional validation using Yoda1 stimulation and calcium imaging confirmed effective Piezo1 KO: WT cells showed strong Piezo1-mediated calcium influx while KO cells remained largely unresponsive, with only occasional weak responses likely due to NHEJ repair events. mCherry fluorescence expression in KO cells confirms transgene expression and successful cell engineering.

Conclusions:
We successfully generated and validated PIEZO1 KO Jurkat cells and CALB2 KO PC3 cells through genomic, proteomic, and functional approaches. With these validated cell lines, our lab now holds valuable research tools for our ongoing investigations on mechanosensitive signaling pathways in immune and cancer systems, enabling studies of T cell mechanotransduction in the context of cancer and calretinin's role in metastasis and mechanical adaptation.

Acknowledgements and/or References (Optional): : Acknowledgements
This work was funded by CPRIT RR230029. This work was supported by the Genetic Design and Engineering Center (GDEC) at Rice University, which is funded by CPRIT RP210116.

References
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