|With the advent of high throughput RNA sequencing technology and higher density tiling arrays, the transcriptional activities across the human genome have been investigated and led to the observation of widespread transcription of the genome [1,2]. While only less than 2% of the mammalian genome codes for proteins, tens of thousands of genomic sites are pervasively transcribed and produce non-coding RNAs, including microRNAs, Piwi-interacting RNAs, small interfering RNAs, small nucleolar RNAs and long non-coding RNAs (lncRNAs). LncRNAs were originally defined as transcribed RNA molecules longer than 200 nucleotides and with little protein-coding potential. Recently, emerging evidences have shown that lncRNAs play important roles in a variety of biological processes including development, differentiation, metabolism, genome regulation and cancer progression [3-5]. Nonetheless, due to the heterogeneity and low abundance, in most cases, most lncRNAs have no genetic evidence to support their in vivo function.
|Gene-targeted knockout technology has provided a powerful tool for elucidating the function of lncRNA genes in vivo, making the connection from mouse to mechanism. For example, Malat1, known as metastasis associated lung adenocarcinoma transcript 1, is among the most abundant and highly conserved lncRNAs. Malat1 was identified as an oncogene that promoted tumorigenesis and found to regulate pre-mRNA splicing in nuclear speckles and promote E2F1 target gene expression during cell cycle progression in vitro [6-9]. Recently, Malat1 knockout models have shown that loss of Malat1 in vivo has no apparent phenotypes and compatible with formation of nuclear speckles [10,11], but alters the transcription of Malat1 neighboring genes and metastasis-associated genes [12,13]. These loss-of-function models further support the critical function of Malat1 as a regulator of gene expression governing hallmarks of lung cancer metastasis. Along with more and more differentially expressed lncRNAs have been unraveled and screened during various physiological processes and disease, understanding the functions of these lncRNA genes require lose-of-function screen by deleting or modifying genes, and followed by studying the resulting phenotypes. More recently, a novel powerful genome-editing tool - the type II prokaryotic CRISPR/Cas system has been successfully employed for genome engineering, thus holding great potential for deciphering the function of lncRNAs in vivo.
|The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas) were originally identified as components of the bacterial adaptive immune response to invading viral and plasmid sequences [14,15]. This CRISPR/Cas system relies on integration of foreign DNA fragments into CRISPR loci. After transcription and processing, these inserts produce a short CRISPR RNA (crRNA), which recognizes a complementary stretch of nucleotides (the protospacer) within foreign DNA and then anneals to a trans-activating CRISPR RNA (tracrRNA), forming ribonucleoprotein complexes with the Cas9 nuclease and generating site-specific double strands breaks in the foreign DNA.
|Generation of a targeted genomic double strands break has been considered as the rate-limiting step in the development of gene targeting technology for genome engineering. By combining crRNA and tracrRNA into a single chimeric guide RNA (gRNA) and expressing it alongside Cas9 endonuclease, several groups have shown that they can engineer the CRISPR/Cas system to function with custom gRNA to efficiently induce double strands break in specific genes in human cells [16-18]. For example, Mali et al.  obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. Cong et al.  applied the CRISPR/Cas system to mediate genomic cleavage as efficiently as a pair of TALE nucleases (TALEN) targeting the same EMX1 element. Cho et al.  showed that CRISPR/Cas system can target two genomic sites and induce indels with frequencies of up to 33%. These three independent groups showed the easy programmability and wide applicability of the CRISPR/Cas technology, establishing the potential of the CRISPR/Cas system for high-throughput applications.
|LncRNAs are marked with their abundance in the genome. It is clear that the majority of disease-associated SNPs are located within non-coding regions and impacts lncRNA expression . Kumar et al.  showed that the associated of SNPs with expression levels of lncRNAs are also associated with complex traits and diseases. Studying the epistatic lncRNAs through CRISPR/Cas system may allow for dissection of phenotypes that are normally masked by compensatory mechanisms. Accordingly it requires the simultaneous introduction of multiple gRNAs into human cells to achieve multiplex lncRNA genes editing of multiple targeted loci. Subsequent studies further support that CRISPR/Cas system can efficiently mediate the simultaneous disruption of five genes in mouse embryonic stem cells, allowing the one-step generation of animals carrying mutations in multiple genes or reporter and conditional alleles [21,22]. Furthermore, the CRISPR/ Cas system also provides the probability for studies of complex genetic disease in a Petri dish using human embryonic stem cells and induced pluripotent stem cells .
|The CRISPR/Cas system may yet have limitations although it undoubtedly possesses great potential for genome editing. For instance, target site selection may be limited by requirement for a dubbed protospace adjacent motif (PAM) sequence. Two current Cas9 nucleases are derived from Streptococcus pyogenes and Streptococcus thermophiles require PAMs of 5’-NGG-3’ and 5’-NNAGAAW-3’ respectively, which may constraint the selection of target sequences . Another important limitation of the CRISPR/Cas system is its potential off-target effects [16-18]. Although each base within the guide RNA sequence contributes to overall specificity, multiple mismatches between the guide RNA and its complementary target DNA sequence can be tolerated depending on the quantity, position and base identify of mismatches. However, Ran et al recently developed a strategy that combines the D10A mutant nickase version of Cas9 from Streptococcus pyogenes (SpCas9n) with a pair of offset gRNAs complementary to opposite strands of the target sites to introduce targeted double strand breaks . This double nicking strategy reduces off-target activity by 50 to 1,500-fold in cell lines and facilitates gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency, thus extensively improving the specificity of Cas9-mediated genome editing.
|In the last decade, the rapid discovery of lncRNAs by highthroughput technologies has advanced our knowledge of transcriptome complexity. LncRNAs have been shown as a novel layer of regulation of diverse physiological activities. Dysregulation of lncRNAs and lncRNA expression-associated SNPs can lead to numerous diseases including cancers. It is inevitable to dissect into lncRNA structure and physiological function by in vivo knockout phenotypes. Timely, the novel CRISPR/Cas technology for genome-editing is moving so fast that it undoubtedly allows the accelerated study of lncRNAs in vivo in the near future.
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