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      Application of CRISPR-Cas9 for Long Noncoding RNA Genes in Cancer Research

      Author(s): 1 , 2 , 1 , 2
      Publication date Created:
      Publication date (Print):
      Journal: Human Gene Therapy
      Publisher: Mary Ann Liebert Inc

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          Abstract

          <p class="first" id="d5034856e71">Long noncoding RNAs (LncRNA), a class of transcripts with lengths &gt;200 nt, play a master role in the regulation of cancer pathogenesis. Recently, the CRISPR-Cas9 system has been explored as a revolutionary genome editing tool for molecular biology. Growing evidence shows that LncRNAs can be targeted by the CRISPR-Cas9 system used for evaluating its function. Thus, the CRISPR-Cas9 systems provide a novel gene-editing strategy for the modification of LncRNA expression. This review summarizes current knowledge of the functions and underlying mechanisms of LncRNA by CRISPR-Cas9. Emerging strategies for non-viral/viral delivery of CRISPR-Cas9 in a clinical context are also discussed. </p>

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              Genome engineering using the CRISPR-Cas9 system.

              F. Ran, Patrick D Hsu, Jason Wright … (2013)
              Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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                Author and article information

                Journal
                Title: Human Gene Therapy
                Abbreviated Title: Human Gene Therapy
                Publisher: Mary Ann Liebert Inc
                ISSN (Print): 1043-0342
                ISSN (Electronic): 1557-7422
                Publication date Created: January 2019
                Publication date (Print): January 2019
                Volume: 30
                Issue: 1
                Pages: 3-9
                Affiliations
                [1 ]Center for Translational Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
                [2 ]Key Laboratory for Tumor Precision Medicine of Shaanxi Province, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
                Article
                DOI: 10.1089/hum.2018.063
                PubMed ID: 30045635
                SO-VID: 98ee50cc-a5ae-4f85-a4b6-69ac5e4986eb
                Copyright © © 2019
                License:

                https://www.liebertpub.com/nv/resources-tools/text-and-data-mining-policy/121/

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