Engineering Success
1.CRISPR-Cas9 is a Promising Therapeutic Approach for Breast Cancer
Breast cancer continues to be a complex and widespread health issue impacting millions of people globally [1]. Today, it remains the leading cancer diagnosis and cause of cancer-related death in females, and is a major contributor to the cancer burden, even when both sexes are combined, representing the second most frequently diagnosed cancer and the fourth most common cause of cancer-related mortality in 2022[2-4].
Triple-negative breast cancer (TNBC) is a subtype of breast cancer. It is a heterogeneous, recurring cancer associated with a high rate of metastasis, poor prognosis, and lack of therapeutic targets. Although target-based therapeutic options are approved for other cancers, only limited therapeutic options are available for TNBC [5]. Recent advancements in biomedical research have greatly improved our understanding of cancer, leading to the investigation of innovative therapeutic approaches for breast cancer. Notably, CRISPR-Cas9 therapy has demonstrated significant promise in cancer treatment.
In our study, TEAD4 was found to be highly expressed in Breast cancer [5]. We designed CRISPR-Cas9 guide RNAs (gRNAs) that specifically target the TEAD4 gene. Transfection of these CRISPR-Cas9 into Breast cancer cell lines resulted in the knockout of TEAD4 expression. As TEAD4 plays a critical role in modulating the downstream Hippo pathway, which is known to promote tumor proliferation, migration, and invasion. we assessed the impact of TEAD4 knockdown on cell proliferation, migration, and invasion in Breast cancer cell lines.
2. gRNA design
2.1 Retrieve the TEAD4 mRNA Sequence
To construct the TEAD4 knockout plasmid, the initial step involves retrieving the TEAD4 gene sequence. This sequence is then used to design gRNA.
The NCBI online database is accessable to search for and download full-length gene sequence (https://www.ncbi.nlm.nih.gov/), in which we search up the key word “TEAD4” (Figure 1A). And then, we download three different transcripts of TEAD4 (Figure 1B). The sequence file is subsequently imported into SnapGene software to analyze, and we can find these transcripts have a common sequences in their exon (Figure 1C). Our final gRNA sequence is designed to target the conserved exonic sequence common to these TEAD4 transcripts.
Figure 1 Retrieve the TEAD4 mRNA Sequence
(A) Search of TEAD4 in the NCBI (B) Choose the TEAD4 transcript (C) The common sequences of different transcripts of TEAD4.
2.2 Design the gRNA Sequence targeted to TEAD4
The first step in constructing gRNA targeted to TEAD4 involves designing a specific sequence that exist in the common sequences of different transcripts of TEAD4.
We designed the gRNA of TEAD4 by an online database (http://www.e-crisp.org/). After we input the TEAD4 gene name and sequence (Figure 2A), the database can output several possible gRNA sequence (Figure 2B). We chose two pairs of gRNA squences with the least off-target possibility.
Figure 2 Design of gRNA Sequences Targeting TEAD4
(A&B) Search for gRNA sequences in database
3. Construction of CRISPR plasmids Targeted to TEAD4
CRISPR-Cas9 technology provides an effective strategy for achieving stable knockout of specific gene expression in eukaryotic cells. In experimental practice, this is accomplished by cloning the gRNA sequence together with CRISPR components into a plasmid vector, which is then transfected into the target cells to facilitate the knockout of the intended gene. To ensure the success of the knockout, we further checked whether the gRNA sequence was present in the CDS of TEAD4, and pick one gRNA sequence. Then, we used CRISPR-Cas9 technology to construct TEAD4-KO1 plasmid.
Following the determination of the gRNA sequence, forward and reverse oligonucleotide primers are designed. These oligonucleotides are chemically synthesized and then annealed to form a double-stranded DNA template with sticky ends compatible with the cloning vector lentiCRISPRv2. The map of the original lentiCRISPRv2 vector was listed below (Figure 3). It can be seen that the BsmBI (Esp3I) restriction sites for gRNA cloning make it fully compatible with Golden Gate Assembly,
Figure 3 Map of lentiCRISPRv2 vector
The sequence and map of lentiCRISPRv2 vector originated from addgene.
The backbone plasmids were cut by restriction endonuclease enzyme BsmBI (Esp3I), then ligated with the gRNA sequence by T4 DNA ligase. After that, this recombinant plasmid will be transformed into the DH5α cell, and cultured in the LB culture medium with Ampicillin, aiming to test whether the gRNA sequences were inserted in the plasmid successfully (Figure 4A). After that, the recombinant plasmids were also confirmed by using Sanger sequencing (Figure 4B). Ultimately, plasmids harboring the intended gRNA sequence was successfully generated and verified.
Figure 4 The TEAD4-KO1 plasmid
(A)Pick up the single clone from solid plate (B) Upper: TEAD4-KO1 sequence validated in the composite plasmid through Sanger Sequencing
4. CRISPR-CAS 9 successfully inhibit TEAD4 Expression
At first, we chose two cell lines for the following experiments, the first is MCF-7 cell, it is the normal breast cancer cell; the second is MDA-MB-231 cell, which is a triple-negative breast cancer cell. We compared the effect of our drug on the two cells to investigate whether it is specific to TNBC treatment.
Western blot analysis was used to assess TEAD4 protein expression. The results demonstrated a significant reduction in TEAD4 protein levels following transfection with shRNA plasmids (Figure 5), confirming the high specificity and effectiveness of our plasmid on the two cell lines (Figure 5).
Figure 5 The detection of TEAD4 knockout efficiency
(TEAD4 protein expression was determined by western blot analysis. Tubulin protein was used for equal loading assessment)
5.1 TEAD4-Targeted plasmid inhibited cell proliferation on the CCK-8 Assay
The Cell Counting Kit-8 (CCK-8) assay is a practical and widely used method to evaluate cellular toxicity and proliferation. The intensity of color development correlates with the rate and extent of cell proliferation.
To further assess proliferative capacity, MCF-7 and MDA-MB-231 were transfected with sh-TEAD4 plasmids at varying dosages (0 µg, 1µg, 2 µg, 4 µg) and detected the OD value at 24 h and 48 h (Figure 6).
Statistical analysis revealed no significant changes in proliferation within the NC group at 24 h after treatment with varying plasmid dosages on MCF-7 cell. At 48 h, only 1µg plasmid group showed significant inhibitory effect compared to control group.
In contrast, sh-TEAD4 plasmid at different concentrations significantly inhibited MDA-MB-231 proliferation, with no notable differences observed between 1 µg, 2 µg and 4 µg treatments at 48 h, suggesting that the inhibition was not dose-dependent.
Figure 6 the proliferation ability after transfected with shRNA targeted to TEAD4
(left is the chromogenic reaction of CCK8 experiment in 96 well plate of two cell lines. Relative cell viability was normalized by the control group ***means p<0.01, ****means p<0.001 by Student’s t-test significance, ns means none significance).
5.2 TEAD4-targeted plasmid inhibited cell migration rate in the scratch assay
Cell migration is a key procedure involved in many biological processes. The scratch assay, measures the migration of cells across a scratch induced gap in vitro.
To further assess migration capacity, MCF-7 and MDA-MB-231 were also transfected with sh-TEAD4 plasmids at varying dosages (0 µg, 1µg, 2 µg, 4 µg).
Statistical analysis revealed decrease of migration among 1 µg, 2 µg and 4 µg treatments compared to NC group of MDA-MB-231. Moreover, the inhibition was dose-dependent (Figure 7).
Figure 7 the migration ability after transfected with shRNA targeted to TEAD4
(Scale bar:5 μm. Quantitative analysis about the percentage of closure. **means p<0.01, ****means p<0.001 by Student’s t-test significance, ns means none significance) .
5.3 shRNA targeted to TEAD4 inhibits cell invasion in transwell Assays
The transwell assay is a useful technique to assess the invasion capability of cells. Two cells were transfected with different plasmids: a negative control (NC), sh-TEAD4. These cells were then treated with varying concentrations of the plasmids 0 µg, 1 µg, 2 µg, and 4 µg.
Statistical analysis revealed that 2 µg and 4 µg group showed significant decrease of cell invasion ability on the MCF-7 cell. Moreover, sh-TEAD4 plasmid at different concentrations significantly inhibited MDA-MB-231 invasion with significant differences between the 1 µg, 2 µg and 4 µg treatments. It suggested that the inhibition was dose-dependent (Figure 8).
Figure 8 Transwell invasion assay
(Scale bar:5 μm. Representative images of the total view of the transwell were shown, MCF-7 and MDA-MB-231 cell were transfected with plasmid, sh-TEAD4 composite plasmid that could silence TEAD4 gene expression, data were collected from 10 fields of three independent experiments. ***means p<0.01, ****means p<0.001 by Student’s t-test significance, ns means none significance.)
We explored the impact of shRNA Targeting TEAD4 on the proliferation, migration, and invasion ability through the CRISPR-CAS9 method. We conducted the experiment on two cell lines, one is MCF-7 cell which stands for the normal breast cancer cell and the other is MDA-MB-231 cell, which stands for triple-negative breast cancer cell.
Our results revealed that TEAD4 knockout significantly reduced cell proliferation, migration and invasion on MDA-MB-231 cell. And the effect is not notable on MCF-7 cell. It indicated that our drug was specific to the TNBC treatment. These findings underscore the therapeutic potential of TEAD4 as a target for targeted treatment in TNBC, which presenting promising opportunities for clinical applications.
References
1. Obeagu, E.I. and G.U. Obeagu, Breast cancer: A review of risk factors and diagnosis. Medicine (Baltimore), 2024. 103(3): p. e36905.
2. Bray, F., et al., Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2024. 74(3): p. 229-263.
3. Giaquinto, A.N., et al., Breast Cancer Statistics, 2022. CA Cancer J Clin, 2022. 72(6): p. 524-541.
4. Loibl, S., et al., Breast cancer. Lancet, 2021. 397(10286): p. 1750-1769.
5. Singh, D.D. and D.K. Yadav, TNBC: Potential Targeting of Multiple Receptors for a Therapeutic Breakthrough, Nanomedicine, and Immunotherapy. Biomedicines, 2021. 9(8).
