VEZF1 knockout in HUVECs cells by TALENs
Strategy modification
To optimize the strategy of Vascular endothelial zinc finger 1 (VEZF1) gene knockout in Human umbilical vein endothelial cells (HUVECs), Transcription activator-like effector nucleases (TALEN) is going to be used instead of CRISPR/Cas9. And validation for the knockout is going to be assessed on different levels including gene level, expression level, and knocked-out VEZF1 protein impact on HUVECs cells.
To begin with, regarding the choice of knockout strategy, Although CRISPR/Cas9 have many advantages over TALENs including its efficiency, ease of construction and low cost (Naeem et al., 2020). However, a significant shortcoming of CRISPR/Cas9 is its off-target mutagenesis. Since CRISPR could modify several locations due to its RNA-DNA recognition, which allows mismatches in the genome, and produces cuts in undesired regions (Ernst et al., 2020). Unlike CRISPR, using TALENs generates more specific targeting by protein-DNA recognition with the transcription activator like (TAL) protein, which have reduced off-target and cytotoxic. (Koo et al., 2015). The main purpose of this experiment is to knockout VEZF1 gene with minimized off-target therefore TALENs would do a better job despite its limitations. Especially since we are less concerned about efficiency in gene knockout.
Knockout design
First, TALEN system is engineered with ChopChop and UCSC genome browser. Two dimers of TALENS each one composed of transcription activator like protein (TAL) as a binding domain with 12-33 repeats that will bind to a targeted sequence in VEZF1 gene, attached to a fokl nuclease which will cleave the targeted sequence and together TALEN dimers will produce a double strand cleavage in the VEZF1 gene (Yoshida & Treen, 2018). TALENs pairs are cloned in two vectors. Then HUVECs cells are transfected by direct injection of TALENs plasmids (Feng et al., 2014). After the gene is cleaved, NHEJ system repairs the cleavage and introduces IN/DEL mutation (Zheng et al., 2020).
Monoclone selection
For selection, the targeted DNA from HUVECs cells is amplified by PCR and then denatured and reannealed to allow are screening by the Mismatch Cleavage Detection Assay using T7 endonuclease 1, which is a commonly used assay that cleaves mismatching reannealed amplicons resulting in two small fragments that can be seen in gel electrophoresis. then monoclone with the desired knock-out modification is isolated.
Knockout validation
For the validation of gene level, gDNA is sequenced by Sanger sequencing to check knockout sequence and compare it to the wildtype. Then, validation on gene expression level is done by quantitative Real-Time PCR (RT-PCR), cDNA is synthesized by reverse transcriptase, and then amplified in PCR with VEZF1, and amplification plot is graphed to compare VEZF1 expression in WT and mutant cells. Finally, the assessment of the impact of VEZF1 silencing on HUVECs cells level is going to be done by tube-formation assay. This assay is chosen because VEZF1 protein is involved in the regulation of angiogenesis and vascular growth in endothelial cells (AlAbdi et al., 2018), which impacts tube-formation (Li et al., 2020). Therefore, examination of disrupted and abnormal tube-formation (tube length and branches) by the tube-formation assay is a good indicator for the lost function of knocked-out VEZF1. This is achieved by seeding WT and mutant HUVECs cells on a gelled matrix and are incubating them to allow tube-formation, and then imaging their tube-formation development over time to examine any abnormalities in the mutant HUVECs.
References
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Feng, Y., Zhang, S., & Huang, X. (2014). A robust TALENs system for highly efficient mammalian genome editing. Scientific Reports, 4(1). https://doi.org/10.1038/srep03632
Koo, T., Lee, J., & Kim, J.-S. (2015). Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9. Molecules and Cells, 38(6), 475–481. https://doi.org/10.14348/molcells.2015.0103
Li, L., Williams, P., Gao, Z., & Wang, Y. (2020). VEZF1–guanine quadruplex DNA interaction regulates alternative polyadenylation and detyrosinase activity of VASH1. Nucleic Acids Research, 48(21). https://doi.org/10.1093/nar/gkaa1092
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020). Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing. Cells, 9(7), 1608. https://doi.org/10.3390/cells9071608
Yoshida, K., & Treen, N. (2018). TALEN-Based Knockout System. Transgenic Ascidians, 131–139. https://doi.org/10.1007/978-981-10-7545-2_12
Zheng, N., Li, L., & Wang, X. (2020). Molecular mechanisms, off‐target activities, and clinical potentials of genome editing systems. Clinical and Translational Medicine, 10(1), 412–426. https://doi.org/10.1002/ctm2.34