Anja Petrović*, Vera Pavić and Marija Stanković
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
anjap5301 [at] gmail.com
Abstract
A human ZBTB14 protein belongs to a large conserved family of transcriptional regulators which are crucial in the regulation of physiological and pathological processes such as development, differentiation, metabolism, apoptosis and carcinoma. Transcription factor ZBTB14 was found to regulate the expression of several genes (e.g. c-MYC, FMR1, DT, IL6 and LIF), and also to be involved in the maintenance of genome stability. So, ZBTB14 transcription factor affects multiple pathways and cellular functions, but its role in the regulation of macrophage development is still largely unknown.
The main objective of this study was to generate ZBTB14 knockout (KO) using U937 monocytes that can be further employed in research of ZBTB14 function.
To generate ZBTB14 KO clone, CRISPR-Cas9 approach was applied in U937 monocytes. Potential U937 ZBTB14KO clones were tested by PCR and Sanger sequencing. Selected U937 ZBTB14KO clone and U937 cells were subjected to Whole Genome Sequencing (WGS) and bioinformatics analysis. DNA library preparation and sequencing were performed by using the MGI technology. Clean reads were aligned to the GRCh38 human reference genome using the Burrows–Wheeler aligner and processed with SAMtools, MANTA and IGV tool. Off-targets were analysed using BLASTn algorithm. The effect of ZBTB14 KO was examined on potential gen-target using RT-PCR and ELISA method.
A homozygous deletion of 12.902 bp was found at the chromosome 18 of the U937 ZBTB14KO clone. The deletion encompassed 5′ UTR with the full promoter, the first exon, intron and a part of the second exon of the ZBTB14 gene, completely preventing the transcription of this gene. Potential off-target sequences showed no changes in the genome of U937 ZBTB14KO clone. Interestingly, the expression and secretion of MMP9 was significantly higher (p=0.0043 and p=0.0031, respectively) in PMA-induced U937 ZBTB14KO macrophages than in U937 macrophages.
Herein, we showed that the application of WGS approach was necessary to successfully confirm the homozygous deletion of 12.902 bp in U937 ZBTB14KO clone. The absence of changes in off-target sequences indicated the precision in design and application of CRISPR-Cas9 approach. Also, we showed a novel role of ZBTB14 transcriptional factor in the regulation of MMP9 in macrophages.
Keywords: CRISPR-Cas9, WGS, ZBTB14, transcription regulation