Strategies for activity analysis of single nucleotide polymorphisms associated with human diseases.

Genome-wide association studies (GWAS) have identified a large number of single nucleotide polymorphism (SNP) sites associated with human diseases. In the annotation of human diseases, especially cancers, SNPs, as an important component of genetic factors, have gained increasing attention. Given that most of the SNPs are located in non-coding regions, the functional verification of these SNPs is a great challenge. The key to functional annotation for risk SNPs is to screen SNPs with regulatory activity from thousands of disease associated-SNPs. In this review, we systematically recapitulate the characteristics and functional roles of SNP sites, discuss three parallel reporter screening strategies in detail based on barcode tag classification, and recommend the common in silico strategies to help supplement the annotation of SNP sites with epigenetic activity analysis, prediction of target genes and trans-acting factors. We hope that this review will contribute to this exuberant research field by providing robust activity analysis strategies that can facilitate the translation of GWAS results into personalized diagnosis and prevention measures for human diseases.

Increasing Genome Editing Efficiency of Cas9 Nucleases by the Simultaneous Use of Transcriptional Activators and Histone Acetyltransferase Activator.

The CRISPR-Cas9 system shows diverse levels of genome editing activities on eukaryotic chromatin, and high-efficiency sgRNA targets are usually desired in application. In this study, we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing. However, the strategy that increases chromatin openness by fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR-Cas9 targets in Lenti-X 293T cells. Under the enlightenment that histone acetylation increases eukaryotic chromatin accessibility, we developed a composite strategy to further improve genome editing by activating histone acetylation. We demonstrate that promoting histone acetylation using the histone acetyltransferase activator YF-2 can improve the genome editing by Cas9 and, more robustly, by the Cas9 transcriptional activator (Cas9-AD). This strategy holds great potential to enhance CRISPR-Cas9 genome editing and to enable broader CRISPR gRNA target choices for experiments in eukaryotes.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection.

Detection of genome editing with quantitative polymerase chain reaction (PCR) primarily relies on and is limited by its ability to discriminate genome modification from the wild-type sequence. An enhanced DNA polymerase variant with superior specificity is needed for this application. Here, we perform semi-rational molecular evolution on full-length Taq polymerase to screen high-specific variants that meet the requirements of gene variation detection. We substituted each of the 40 polar amino acids in direct contact with the primer/template duplex and conducted extensive random mutagenesis to generate a Taq mutation library. Screening on a quantitative PCR system with insertion and deletion-containing templates identified a series of improved Taq variants. We demonstrate that the Taq388 variant bearing three amino acid substitutions, S577A, W645R, and I707V, has improved sensitivity to insertion and deletion-derived primer/template mismatch by a ΔCt value of 25-26 and is superior for application in evaluating CRISPR-Cas9 editing efficiency and single-cell clone genotyping. In addition, the Taq variant shows substantial potential for single-nucleotide polymorphism detection by means of allele-specific PCR because of its high sensitivity to mismatches.

Dinucleotide tag-based parallel reporter gene assay method enables efficient identification of regulatory mutations.

BACKGROUND: The causal single nucleotide polymorphisms (SNPs) leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely relies on the parallel reporter gene assay system. However, the common DNA barcodes used in parallel reporter gene assay systems typically because nucleotide composition bias, and many barcodes must be allocated for each sequence to reduce the bias effect. MAIN METHODS AND MAJOR RESULTS: Here, a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on next-generation sequencing is described. The DiR system is more robust than the classical luciferase assay method, particularly for the investigation of moderate-level regulatory elements. The authors applied the DiR-seq assay in the functional evaluation of SNPs with prostate cancer risk and nominated two and six regulatory SNPs in PC-3 and LNCaP cells, respectively. CONCLUSIONS AND IMPLICATIONS: The DiR system has great potential to advance the functional study of SNPs associated with polygenic disease risks.

Functional Screenings Identify Regulatory Variants Associated with Breast Cancer Susceptibility.

Genome-wide association studies (GWAS) have identified more than 2000 single nucleotide polymorphisms (SNPs) associated with breast cancer susceptibility, most of which are located in the non-coding region. However, the causal SNPs functioning as gene regulatory elements still remain largely undisclosed. Here, we applied a Dinucleotide Parallel Reporter sequencing (DiR-seq) assay to evaluate 288 breast cancer risk SNPs in nine different breast cancer cell lines. Further multi-omics analysis with the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing), DNase-seq (DNase I hypersensitive sites sequencing) and histone modification ChIP-seq (Chromatin Immunoprecipitation sequencing) nominated seven functional SNPs in breast cancer cells. Functional investigations show that rs4808611 affects breast cancer progression by altering the gene expression of NR2F6. For the other site, rs2236007, the alteration promotes the binding of the suppressive transcription factor EGR1 and results in the downregulation of PAX9 expression. The downregulated expression of PAX9 causes cancer malignancies and is associated with the poor prognosis of breast cancer patients. Our findings contribute to defining the functional risk SNPs and the related genes for breast cancer risk prediction.

rs10514231 Leads to Breast Cancer Predisposition by Altering ATP6AP1L Gene Expression.

Numerous genetic variants located in autophagy-related genes have been identified for association with various cancer risks, but the biological mechanisms underlying these associations remain largely unknown. Here we investigated their regulatory activity with a parallel reporter gene assay system in breast cancer cells and identified multiple regulatory SNP sites, including rs10514231. It was located in the second intron of ATG10 and showed gene regulatory activity in most breast cancer cells we used. Mechanistically, the T allele of rs10514231 led to ATP6AP1L downregulation by decreasing the binding affinity of TCF7L2. Overexpression of the ATP6AP1L gene in cancer cells diminished cell proliferation, migration, and invasion. Notably, ATP6AP1L downregulation correlated with breast cancer risk and with poor prognosis in patients. These results provide a plausible mechanism behind the association of rs10514231 with breast cancer risk and will be important for more effective therapeutic target identification for precision medicine.

Parallel Reporter Assays Identify Altered Regulatory Role of rs684232 in Leading to Prostate Cancer Predisposition.

Functional characterization of cancer risk-associated single nucleotide polymorphism (SNP) identified by genome-wide association studies (GWAS) has become a big challenge. To identify the regulatory risk SNPs that can lead to transcriptional misregulation, we performed parallel reporter gene assays with both alleles of 213 prostate cancer risk-associated GWAS SNPs in 22Rv1 cells. We disclosed 32 regulatory SNPs that exhibited different regulatory activities with two alleles. For one of the regulatory SNPs, rs684232, we found that the variation altered chromatin binding of transcription factor FOXA1 on the DNA region and led to aberrant gene expression of VPS53, FAM57A, and GEMIN4, which play vital roles in prostate cancer malignancy. Our findings reveal the roles and underlying mechanism of rs684232 in prostate cancer progression and hold great promise in benefiting prostate cancer patients with prognostic prediction and target therapies.

A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells.

CRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3' end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

Deletion of Brg1 causes abnormal hair cell planer polarity, hair cell anchorage, and scar formation in mouse cochlea.

Hair cells (HCs) are mechanosensors that play crucial roles in perceiving sound, acceleration, and fluid motion. The precise architecture of the auditory epithelium and its repair after HC loss is indispensable to the function of organ of Corti (OC). In this study, we showed that Brg1 was highly expressed in auditory HCs. Specific deletion of Brg1 in postnatal HCs resulted in rapid HC degeneration and profound deafness in mice. Further experiments showed that cell-intrinsic polarity of HCs was abolished, docking of outer hair cells (OHCs) by Deiter's cells (DCs) failed, and scar formation in the reticular lamina was deficient. We demonstrated that Brg1 ablation disrupted the Gαi/Insc/LGN and aPKC asymmetric distributions, without overt effects on the core planer cell polarity (PCP) pathway. We also demonstrated that Brg1-deficient HCs underwent apoptosis, and that leakage in the reticular lamina caused by deficient scar formation shifted the mode of OHC death from apoptosis to necrosis. Together, these data demonstrated a requirement for Brg1 activity in HC development and suggested a role for Brg1 in the proper cellular structure formation of HCs.