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

Kabin Xie, Bastian Minkenberg, Yinong Yang

Research output: Contribution to journalArticle

301 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3570-3575
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number11
DOIs
StatePublished - Mar 17 2015

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Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Transfer RNA
Synthetic Genes
Endonucleases
Genetically Modified Plants
RNA Precursors
Genome
Equipment and Supplies
DNA
Research
Genes
Proteins

All Science Journal Classification (ASJC) codes

  • General

Cite this

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abstract = "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.",
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Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. / Xie, Kabin; Minkenberg, Bastian; Yang, Yinong.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 11, 17.03.2015, p. 3570-3575.

Research output: Contribution to journalArticle

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