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Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Proceedings of the National Academy of Sciences - PNAS, 2015-03, Vol.112 (11), p.3570-3575 [Peer Reviewed Journal]

Volumes 1–89 and 106–112, copyright as a collective work only; author(s) retains copyright to individual articles ;Copyright National Academy of Sciences Mar 17, 2015 ;ISSN: 0027-8424 ;EISSN: 1091-6490 ;DOI: 10.1073/pnas.1420294112 ;PMID: 25733849

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  • Title:
    Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system
  • Author: Xie, Kabin ; Minkenberg, Bastian ; Yang, Yinong
  • Subjects: Base Sequence ; Biological Sciences ; CRISPR-Cas Systems ; Gene expression ; Genes ; Genes, Plant ; Genetic Engineering ; Genomes ; Molecular Sequence Data ; Mutagenesis - genetics ; Mutation - genetics ; Oryza - genetics ; Plants, Genetically Modified ; Protoplasts - metabolism ; RNA Editing - genetics ; RNA Processing, Post-Transcriptional - genetics ; RNA, Guide, CRISPR-Cas Systems - genetics ; RNA, Transfer - genetics ; Transfer RNA
  • Is Part Of: Proceedings of the National Academy of Sciences - PNAS, 2015-03, Vol.112 (11), p.3570-3575
  • Description: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. Significance The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has recently emerged as an efficient and versatile tool for genome editing in various organisms. However, its targeting capability and multiplex editing efficiency are often limited by the guide RNA (gRNA)-expressing device. This study demonstrates a general strategy and platform for precise processing and efficient production of numerous gRNAs in vivo from a synthetic polycistronic gene via the endogenous tRNA-processing system. This strategy is shown to significantly increase CRISPR/Cas9 multiplex editing capability and efficiency in plants and is expected to have broad applications for small RNA expression and genome engineering.
  • Publisher: United States: National Academy of Sciences
  • Language: English
  • Identifier: ISSN: 0027-8424
    EISSN: 1091-6490
    DOI: 10.1073/pnas.1420294112
    PMID: 25733849
  • Source: GFMER Free Medical Journals
    MEDLINE
    PubMed Central
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