Induced Pluripotent Stem Cells (iPSCs) are reprogrammed cells created from somatic cells. Our lab has pioneered the iPSC technology by inserting four unique transcription factors - Oct3/4, Sox2, Klf4 and Myc that convert somatic cells into pluripotent stem cells1. iPSCs serve as an excellent model for investigating human cell biology and hold a key solution for regenerative medicine because of their potential to replenish diseased or damaged cell types including heart, neurons, liver, pancreas and more 1,2. Subsequently, several studies have been published globally on iPSCs with wide range applications from therapeutic to pharmaceutical science.
Although the molecular function and phenotypic roles of many human proteins have been discovered, we are still in early stages in terms of understanding the roles of several key factors. We can delineate the physiological relevance of these understudied proteins, by modulating their expression levels in their native context. iPSCs are patient-specific reprogrammed cells that can self-renew indefinitely as well as capable of generating all three germ-layers in vitro1,3. iPSCs therefore are an excellent model system for investigating and studying the role of various proteins of interest especially in the context of cell proliferation, differentiation and disease modelling4.
Paraspeckles have been recently discovered as mammalian specific nuclear bodies5. They are comprised of a long, non-coding RNA (nuclear enriched abundant transcript 1-NEAT1) as well as a handful of key proteins belonging to the RNA binding protein of Drosophila behaviour and human splicing (DBHS) family that assemble them. Paraspeckles are found in almost all of mammalian cells but their function is not yet determined. It has recently shown that they might play a significant role in biological process including cellular differentiation and stress response5.
Splicing Factor Proline and Glutamine Rich (SFPQ) is a conserved and core component of paraspeckles. It is an integral protein that helps with the formation and maintenance of paraspeckles in mammalian cells5. SFPQ is also speculated to play a vital role in other biological processes such as DNA repair, RNA splicing and transport6-9. In another study in mice, knocking out of the gene that encodes Nuclear Enriched Abundant Transcript 1 (NEAT1) RNA, another core component of paraspeckles, resultant in a complete loss of paraspeckles, but no apparent phenotypic changes were observed in mice 5. Although the loss of SFPQ, has been shown to result in the loss of paraspeckles in other mammalian organisms, the phenotype of the loss of paraspeckles in human cells is unknown. In the present study, we have investigated the role of paraspeckles in human cells by CRISPR interference based knocking down of SFPQ in iPSCs.
In order to knock down SFPQ gene function in iPSCs, we have used the type II CRISPR-Cas9 system. Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated proteins (Cas) are RNA-mediated adaptive immune system in bacteria and archaea that prevents infection from viruses10,11. The CRISPR-Cas9 technology is a rapid, simple, efficient, cost effective technology used in many organisms for targeted genome editing12-18. It has also been used for correcting mutation in genes for restoring gene functions19,20 such as hemoglobin (HbA) gene21,22, used for epigenetic modifications23,24, for down regulating gene function17, 25-29 and many more applications.
CRISPR interference (CRISPRi) was developed by mutating two active regions of Cas9 (RuvC (D10A) and HNH (H840A) producing catalytically dead or dCas925. dCas9 is catalytically inactive in cleaving the DNA but retains the binding ability to bind DNA in the presence of a Protospacer Adjacent Motif (PAM) and complementary small guide RNA (sgRNA). CRISPRi has been shown to mediate 1000-fold repression in prokaryotes without any noticeable off target effects. Further optimization involves changing the location of sgRNA for improving downregulation25 as well as fusing transcriptional repressor domains including KRAB (Krüppel associated box) domain of Kox1, CS (Chromo Shadow) domain of HP1α, and WRPW domain of Hes130-32. In one relevant study, Qi et al25 employed dCas9 fused with KRAB to transfect into HEK293 cells that expressed GFP being chromosomally integrated. They observed a 5-fold repression of GFP expression. Similarly, Gilbert et al28 have fused dCas9 with dCas9-CS or dCas9-WRPW and found 2-fold repression of GFP. When they have designed and constructed multiple sgRNAs, they found a combination of six sgRNAs can result in a 15-fold GFP repression. CRISPRi has also been shown to be effectively functional in human iPSCs33. It has been further used for generating genome-wide sgRNA libraries that are currently available for designing sgRNAs targeting various human genes34. In our current study, we designed and built sgRNAs for down regulating SFPQ gene expression and found that the loss of SFPQ gene expression results in a severe cell-death phenotype in iPSCs.