Long non-coding RNAs (long ncRNAs, LncRNA) are non-protein coding transcripts longer than 200 nucleotides. Large-scale sequencing of cDNA libraries by NGS indicates that long noncoding RNAs number in the order of tens of thousands in mammals. As of January 2016, 141,353 LncRNA transcripts in human have been annotated in NONCODE. Growing evidence suggests that they are involved in a surprisingly wide variety of cellular functions, including regulation of gene transcription, post-transcriptional regulation and epigenetic regulation. However, only a relatively small proportion (less than two hundred) has been demonstrated to be biologically relevant. Therefore, having a better understanding of the functional roles of LncRNAs would advance our understanding of the complex cellular regulatory network.
Unlike protein coding genes, which have to conserve the codon usage and prevent frameshift mutations in a single long ORF, selection may conserve only short regions of LncRNAs that are constrained by structure or sequence-specific interactions. Therefore, LncRNA knock-out is achieved by deleting large fragment of those conserved sequences instead of short indels for mRNA knock-out.
A large fragment of conserved sequences in a LncRNA is deleted through two nuclease digestion reactions mediated by gRNA-guided Cas9, which are followed by NEEJ repairing.
SyngenTech provides one-stop service for generation of gene knock-out cell lines or model animals using CRISPR/Cas9 technology, including the upstream gRNA design and CRISPR/Cas9 vector construction to the downstream genotyping and breeding to homozygosis. You can also outsource parts of this complex process to us. We will offer you custom and top-quality services at unbeatable prices.
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