Evaluating Basigin promoter variants by CRISPR-Cas9 genome editing

Abstract

Background: Sequence variation within transcriptional factor binding sites can influence gene expression. We have found a four-nucleotide genetic variant in the Basigin (BSG) promoter region that disrupts a predicted GATA-1 binding site. BSG encodes a red blood cells surface protein on that is exploited as an invasion receptor by the malaria parasite Plasmodium falciparum. Hence the identified variant could mediate resistance, which is common in West Africa
Aims and Objective: The aim of the study was to test the functional relevance of the GATA polymorphism and evaluate the effect of variant on the transcription of BSG by CRISPR-Cas9 genome editing as well as genotyping and real time PCR of samples of African origin to correlate BSG expression and protein levels. Furthermore to study the binding of GATA-1 transcription factor at reference/variant DNA sequences by electrophoretic mobility shift assay (EMSA).
Methods and Results: EMSA was performed by incubating nuclear extract from K562 cells with reference sequence (i.e., promoter containing the identified GATA motif) and variant sequence (i.e., promoter lacking the GATA motif) probes followed by separation on gel, the EMSA assay showed stronger binding to the reference site than the variant. Additionally the genome editing CRISPR-Cas9 technique was employed to create cuts in the promoter near the site of the variant-of-interest. The Cas9-sgRNA vector successfully cut DNA and repaired by non-homologous end joining (NHEJ) as verified by Sanger sequencing. Finally we analyzed blood samples from African American blood donors by directed genotyping (Taqman assay) and qPCR to test for correlation between the variants and BSG expression. The genotyping data gave a minor allele frequency (MAF) of 2.1%, however in the 1000 Genomes database, the variant is found in 45 out of 1322 alleles (MAF = 3.4%) in the African super population a frequency of 0.02% of the variant allele. And preliminary qPCR amplification results showed lower transcript expression for homozygous sample than heterozygous.
Conclusion: The EMSA showed differential binding to the reference and variant sites. We created a cutting vector to allow edits of the identified region in human cells. The calculated frequency for the deletion variant found to be slightly lower found in thousand genome database. Preliminary qPCR need some more optimization and further findings to understand the correlation between BSG expression and variant.
Keywords: CRISPR, Basigin, GATA-1, Cas9, Plasmodium falciparum

Popular Abstract

Genetic variation within transcription factor binding sites can effect gene expression. For example, variants (i.e. variation in a single or a few nucleotides across a given population) that interrupt binding sites of transcription factors can lead to decreased gene expression, or even null phenotypes where the gene is not expressed at all. We found a variant in Basigin (BSG) gene promoter region, which could affect the binding of GATA-1, a transcription factor essential for the development of red blood cells.
BSG is a protein present on red blood cells that is hijacked as an invasive receptor by the malaria parasite Plasmodium falciparum. The found variant is present in malaria-endemic regions of West Africa and we hypothesise that it confers some form of resistance against malaria infection by decreasing expression of BSG.
In this project we explored the functional effects of the BSG variant by CRISPR-Cas9 genome editing technology. In specific we aimed to insert the variant into a cell line by CRISPR and then test the expression levels by qPCR and western blotting. We are currently creating a cell line carrying the variant on both alleles and to do so we successfully created a cut in the BSG gene near the variant site by CRISPR-Cas9. The next step is to insert a donor template (carrying the variant) into the target DNA sequence, giving the desired cell line. Once the cell line is created, we will examine BSG expression in these cells. We also tested the association between the BSG expression and the variant in vivo by analyzing blood samples from 215 Africans American donors. We found 9 samples carrying the variant on one allele, but unfortunately did not find any samples carrying deletion on both alleles, making BSG gene expression in vivo difficult.
Theoretically, clarifying the functional effect(s) of this variant will increase our understanding towards resistance to malaria by genetic variation.

• author: Pimpalwar, Neha
• supervisor: Björn Nilsson
• organization: Department of Chemistry
• course: KEMT30 20151
• year: 2015
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: CRISPR, Basigin, Cas9, Plasmodium falciparum, genotyping, EMSA, q-PCR, proteinvetenskap, protein science
• language: English
• id: 7440629
• date added to LUP: 2015-07-02 15:31:46
• date last changed: 2015-07-02 15:31:46