Dissecting the genetic architecture of coronary artery disease by genome engineering

One of the greatest challenges facing biomedical research since the sequencing of the human genome has been to understand the role of genetic variation in human disease. Many genetic variants have been associated with common diseases. However, determining the functional consequences of these variants has been hard. Several variants are often inherited together in tightly linked blocks, making it difficult to determine the causative variant. People have millions of other genetic differences, making it difficult to correlate cellular phenotypes with a particular variant. Different gene sets are expressed in different cells, but it is difficult to extract disease-relevant cells from large numbers of patients. We describe a method with the potential to revolutionize the functional analysis of genetic variation, using custom nucleases to genetically modify individual variants in induced pluripotent stem cells. This process would provide unprecedented analytical power, present the first general method to determine if a variant is causative, and analyze function disease-relevant cell types. We will focus on variants at the 9p21 region of the genome that have been associated with coronary artery disease (CAD). The methods should provide a new way to unlock the wealth of data from genome-wide association studies, and to probe the genetic architecture of common diseases. We will describe our improved methods for inexpensive and rapid construction of highly active zinc finger and TALE nucleases to examine the functional role of polymorphisms at the 9p21 CAD risk locus

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3' and 5' end of miR130a were cloned into pSpCas9(BB)-2AGFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 ⁰C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level

Genome-wide identification of Aedes albopictus long noncoding RNAs and their association with dengue and Zika virus infection.

The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus

Suppression of MiR130a-3p Using CRISPR/Cas9 Induces Proliferation and Migration of Non-Small Cell Lung Cancer Cell Line

BACKGROUND: Molecular alterations of microRNA130a (miR130a) are observed in many types of cancers, including non-small cell lung cancer (NSCLC). However, the role of miR130a in NSCLC has been poorly studied.METHODS: In this study, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 was utilised to knockdown miR130a. The gRNA was designed to target the stem loop, 3’ and 5’ sites of miR130a and stably expressed in A549 cells. Post-treatment, mature levels of miR130a-3p and 5p were quantified, and proliferation and migration assays were conducted.RESULTS: Result showed significant suppression of miR130a-3p and -5p by two and three-fold respectively, when the CRISPR/Cas9 targeted at the 3’ site and stem loop of the miR130a gene. Suppression of miR130a-3p significantly increased the growth and migration of A549 cells, but no significant changes were observed in cells with suppressed expression of miR130a-5p.CONCLUSION: Our encouraging results highlight that the suppression of the miR130a is achievable using CRISPR/Cas9, and suppression of the miR-130a-3p could play an important role in the regulation of NSCLC.KEYWORDS: miR130a, CRISPR-Cas9, non-small cell lung cance