Validation of prostate cancer risk variants rs10993994 and rs7098889 by CRISPR/Cas9 mediated genome editing

Abstract

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the β-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded β-MSP in prostate cancer pathogenesis.

Keywords: Allelic imbalance in expression; CRISPR/Cas9; Enhancers; Prostate cancer; SNP.

Fig 1.

Position of prostate-specific enhancer chr10:51544405–51544819 defined by FANTOM5 project.

Fig 2.. CRISPR/Cas9 mediated deletion of 191bp region flanking rs7098889 leads to significant increase of MSMB expression.

(A) Sanger sequencing showed both LNCaP and AGS cells are heterozygous (C/T) at rs7098889 site. (B) CRISPR/Cas9 mediated rs7098889 deletion was created by paired guide RNAs (rs7098889-g1 and -g4) transfection followed by puromycin selection. The deletion was confirmed by PCR amplification with primer pair (rs7098889-For1 and -Rev1) flanking the deleted region, PCR product runs at 178bp on agarose gel with deletion, and at 369bp without deletion. (C) Real time qPCR showed 9.5 folds MSMB over-expression in prostate cancer LNCaP cells with bulk transfection but not the gastric cancer AGS cells. The expression of downstream NCOA4 gene is barely affected. Error bars represent standard deviation. T-test were performed for 3 independent transfections and p-values are labeled for each pair of comparison. (D) Western blot showed that the MSMB protein product β-MSP is significantly up-regulated in LNCaP cells with deletion, but not in AGS cells. (E) ELISA assay showed that the secreting β-MSP level significantly up-regulated in LNCaP cells with deletion either in the presence (28.0 ng/ml) or absence (2.8ng/ml) of FBS in cell culture.

Fig 3.. Single clone screening of LNCaP cells with rs7098889 deletion results in allelic and dramatic MSMB over-expression.

(A) Transfection titration generated LNCaP bulk cells with lower deletion efficiency for better isolating heterozygous single clones. (B) Illustration of single clone genotypes with homozygous (del/del) and heterozygous deletion (T/del and C/del). (C) Real time qPCR showed dramatic MSMB over-expression (262 and 286 folds) in two (clone 4, 5) out of three clones with rs7098889 T allele (clone 3, 4, 5) but not the C allele (clone 6, 7). Bulk deletion with lower deletion efficiency, thus more heterozygous alleles, generates 15 folds over-expression (lane 3) compared to 9.55 folds from previous experiment (Fig. 1C).

Fig 4.. Allelic expression of MSMB in LNCaP cells.

(A) Demonstration of 360A/T single nucleotide variant (SNV) located in the last exon of MSMB gene. (B) Transcripts from all the MSMB high expressing clones (clone 2, 4, 5) and the LNCaP bulk deletion came from the 360T allele examined by PCR followed by Sanger sequencing of the last exon of MSMB gene flanking the 360A/T heterozygous site.

Fig 5.. CRISPR/Cas9 mediated deletion of 205bp rs10993994 flanking region leads to significant increase of MSMB expression.

(A) Paired gRNA (rs10993994-g4a and -g4b) mediated CRISPR/Cas9 deletion of rs10993994 region was confirmed by PCR amplification with flanking primer pair rs10993994-F1 and -R1. PCR product runs at 274bp on agarose gel with deletion, and at 479bp without deletion. (B) Real time qPCR showed 2.8 folds MSMB over-expression in prostate cancer LNCaP cells with bulk deletion but not in the gastric cancer AGS cells. The expression of downstream NCOA4 gene is down-regulated. (C) Real time qPCR of single clones generated from above bulk transfection. MSMB over-expression was seen in two (clone 5, 6) out of the three clones (clone 4, 5, 6) with rs10993994 C allele (C/del) but not the T allele (T/del, clone 2, 3, 4).