Most human genomes can be transcribed into RNA, but only a few regions produce protein-coding mRNA, while the remaining regions are transcribed into non-coding RNA. Long non-coding RNAs (lncRNAs) are known for their important regulatory roles in a variety of biological processes, such as imprinting, dose compensation, transcriptional regulation, and splicing. In the past 30 years, through genome editing of pluripotent stem cells (PSC), the physiological functions of protein-coding genes have been extensively characterized. However, the research on lncRNAs using genome editing technology has only received attention. In recent years, thousands of lncRNAs have been identified. Some of them have proven to play an important role in PSC. However, advances in genome editing technology have only just begun to be widely used in PSC to study the function of lncRNA. Considering the different functions of lncRNA genomic loci and their transcripts, a variety of genome editing methods should be used to distinguish the functions of lncRNA transcripts and their loci in PSC. lncRNAs are important biomarkers of embryonic development and disease progression. The establishment of the lncRNA reporter gene in the body will enable the monitoring of these processes. The development of emerging CRISPR genome editing technology has opened new doors for lncRNA biology in PSC. Future research should adopt these new strategies to explore the function of lncRNA in PSC. These genome editing tools should also be used to explore the physiological functions of lncRNA in the system. lncRNA knockout Molecular scissors have been used to generate point mutations in key domains of protein-coding genes, which in turn induce premature termination of translation to knock out genes. Unlike protein-coding genes, the transcriptional functional domains of most lncRNAs are still unclear; therefore, it is impossible to study lncRNAs through loss-of-function mutagenesis. Therefore, to knock out lncRNA, it is necessary to completely or partially delete the lncRNA gene. In order to avoid the indirect effects of lncRNA knockout, researchers need to manipulate lncRNA genomic loci without affecting the genomic characteristics of other genes. This, however, is difficult to achieve in some cases. For lncRNAs located in the promoter regions of other genes or overlapping with the exons of protein-coding genes, partial deletion of them through genome editing can only be applied when the expression of other genes is not affected. For the lncRNA in the intron of the protein-coding gene, it is necessary to delete the lncRNA gene without interfering with the splicing of the intron region. The lncRNA in the intergenic region is far away from other genes and can be easily removed by genome editing technology in a manner similar to protein-coding genes. However, intergenic lncRNA loci that overlap with enhancers, such as enhancer RNA, are also difficult to study with genome editing, because the deletion of these sites may interfere with enhancer function and affect the expression of distant genes. With the discovery of more lncRNAs and the further understanding of the function of lncRNAs, the knockout of lncRNAs based on homologous recombination is more widely used to delete lncRNAs in PSCs. In one of the studies, 18 lncRNA genes in mouse ESC were knocked out to produce lncRNA knockout mice. Replacing the lncRNA locus with the lacZ reporter gene allows visualization of the spatiotemporal expression patterns of these lncRNAs in animal models. Another similar study created knockout ESC lines for 20 lncRNAs through gene targeting, and used these ESCs to create knockout mice to study the broad role of lncRNA in mice. These lncRNA knockout mice constitute a valuable supplement to resources for studying the physiological role of lncRNA. Genome editing strategies A single method is not enough to identify all possible functions of lncRNA. A variety of genome editing strategies need to be adopted to discover the function of lncRNA and its transcription site in PSC. Since CRISPR/Cas9-based genome editing technology can effectively delete large fragments of the genome, it has been used to perform genome-wide screening of lncRNA functions. Use multiple gRNAs against a single lncRNA to achieve effective ablation of lncRNA gene expression; therefore, the paired gRNA library can only target hundreds of lncRNAs. Using this method, lncRNAs, which are critical to the survival of cancer cells, have been identified. Another way to use CRISPR-Cas9 to regulate lncRNA is through CRISPR interference (CRISPRi), which is a fusion of inactivated Cas9 (dCas9) and transcription inhibitors (such as KRAB). By recruiting dCas9-KRAB to the promoter region of lncRNA with multiple gRNAs, the expression of lncRNA is hindered by transcription repressors recruited by KRAB protein. CRISPRi was used to manipulate the expression of lncRNA (GAS5, H19, MALAT1, NEAT1, TERC and XIST) in K562 cells.
Written by Creative Biogene
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