Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing

The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers’ ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

[1]  Tessa G. Montague,et al.  Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs , 2014, PloS one.

[2]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[3]  George M. Church,et al.  Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 , 2013, Nature Biotechnology.

[4]  H. Puchta,et al.  Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. , 2014, The Plant journal : for cell and molecular biology.

[5]  Jun Li,et al.  Targeted genome modification of crop plants using a CRISPR-Cas system , 2013, Nature Biotechnology.

[6]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[7]  Kabin Xie,et al.  RNA-guided genome editing in plants using a CRISPR-Cas system. , 2013, Molecular plant.

[8]  Kabin Xie,et al.  Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system , 2015, Proceedings of the National Academy of Sciences.

[9]  M. Spalding,et al.  Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice , 2014, Nucleic acids research.

[10]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[11]  Jian‐Kang Zhu,et al.  Application of the CRISPR-Cas system for efficient genome engineering in plants. , 2013, Molecular plant.

[12]  Daniel F. Voytas,et al.  A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN] , 2015, Plant Physiology.

[13]  Daniel F Voytas,et al.  Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System. , 2015, Molecular plant.

[14]  Xingliang Ma,et al.  Rapid Decoding of Sequence-Specific Nuclease-Induced Heterozygous and Biallelic Mutations by Direct Sequencing of PCR Products. , 2015, Molecular plant.

[15]  Kabin Xie,et al.  Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. , 2014, Molecular plant.

[16]  Yang Lei,et al.  CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. , 2014, Molecular plant.

[17]  Hui-Li Xing,et al.  A CRISPR/Cas9 toolkit for multiplex genome editing in plants , 2014, BMC Plant Biology.

[18]  Clifford A. Meyer,et al.  Sequence determinants of improved CRISPR sgRNA design , 2015, Genome research.

[19]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[20]  Zhongsen Li,et al.  Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN] , 2015, Plant Physiology.

[21]  Botao Zhang,et al.  Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis , 2014, Proceedings of the National Academy of Sciences.

[22]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[23]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[24]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[25]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[26]  W. Filipowicz,et al.  U6 snRNA genes of Arabidopsis are transcribed by RNA polymerase III but contain the same two upstream promoter elements as RNA polymerase II-transcribed U-snRNA genes. , 1990, Nucleic acids research.

[27]  Ya Guo,et al.  Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9 , 2015, Journal of molecular cell biology.

[28]  T. Komari,et al.  Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. , 1994, The Plant journal : for cell and molecular biology.

[29]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[30]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

[31]  Wei Liu,et al.  A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. , 2015, Molecular plant.

[32]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

[33]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[34]  Z. Lippman,et al.  Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System1 , 2014, Plant Physiology.

[35]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.