Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants

CRISPR/Cas9 gene-editing technologies have been very effective in editing target genes in all major crop plants and offer unprecedented potentials in crop improvement. A major challenge in using CRISPR gene-editing technology for agricultural applications is that the target gene-edited crop plants need to be transgene free to maintain trait stability and to gain regulatory approval for commercial production. In this article, we present various strategies for generating transgene-free and target gene-edited crop plants. The CRISPR transgenes can be removed by genetic segregation if the crop plants are reproduced sexually. Marker-assisted tracking and eliminating transgenes greatly decrease the time and labor needed for identifying the ideal transgene-free plants. Transgenes can be programed to undergo self-elimination when CRISPR genes and suicide genes are sequentially activated, greatly accelerating the isolation of transgene-free and target gene-edited plants. Transgene-free plants can also be generated using approaches that are considered non-transgenic such as ribonucleoprotein transfection, transient expression of transgenes without DNA integration, and nano-biotechnology. Here, we discuss the advantages and disadvantages of the various strategies in generating transgene-free plants and provide guidance for adopting the best strategies in editing a crop plant.

[1]  Yanpeng Wang,et al.  Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes , 2017, Nature Communications.

[2]  Martin J. Aryee,et al.  In vivo CRISPR editing with no detectable genome-wide off-target mutations , 2018, Nature.

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

[4]  Fang-Jie Zhao,et al.  Engineering Crops without Genome Integration Using Nanotechnology. , 2019, Trends in plant science.

[5]  I. Baldwin,et al.  Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants , 2013, BMC Plant Biology.

[6]  Soon Il Kwon,et al.  DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins , 2015, Nature Biotechnology.

[7]  Xin Zhang,et al.  Targeted mutagenesis in rice using CRISPR-Cas system , 2013, Cell Research.

[8]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[9]  Bing Yang,et al.  Genome editing in grass plants , 2019, aBIOTECH.

[10]  Shouling Xu,et al.  CRISPR‐S: an active interference element for a rapid and inexpensive selection of genome‐edited, transgene‐free rice plants , 2017, Plant biotechnology journal.

[11]  Enzo Lombi,et al.  Nanotechnology: A New Opportunity in Plant Sciences. , 2016, Trends in plant science.

[12]  Off-target effects and the solution , 2019, Nature Plants.

[13]  J. Sun,et al.  Rice embryo globulins: amino-terminal amino acid sequences, cDNA cloning and expression. , 1996, Plant & cell physiology.

[14]  Q. Qian,et al.  LAZY1 controls rice shoot gravitropism through regulating polar auxin transport , 2007, Cell Research.

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

[16]  G. Cullot,et al.  CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations , 2019, Nature Communications.

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

[18]  R. Wu,et al.  Isolation of an efficient actin promoter for use in rice transformation. , 1990, The Plant cell.

[19]  C. Mariani,et al.  Induction of male sterility in plants by a chimaeric ribonuclease gene , 1990, Nature.

[20]  C. Sparks,et al.  Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. , 2001, Journal of experimental botany.

[21]  J. Šamaj,et al.  Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. , 2015, Biotechnology advances.

[22]  Roger L. Chang,et al.  High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants , 2017, bioRxiv.

[23]  Fredrik Lanner,et al.  Biotechnology: At the heart of gene edits in human embryos , 2017, Nature.

[24]  Ewen Callaway,et al.  CRISPR plants now subject to tough GM laws in European Union , 2018, Nature.

[25]  R. Hartley,et al.  Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease. , 1988, Journal of molecular biology.

[26]  Tingting Gu,et al.  A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants , 2018, Horticulture Research.

[27]  Wang Lihao,et al.  Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants , 2019, Rice Science.

[28]  Ilaria Sanzari,et al.  Nanotechnology in Plant Science: To Make a Long Story Short , 2019, Front. Bioeng. Biotechnol..

[29]  Ru Zhang,et al.  Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration , 2018, Plant biotechnology journal.

[30]  Rongchen Wang,et al.  On Improving CRISPR for Editing Plant Genes: Ribozyme-Mediated Guide RNA Production and Fluorescence-Based Technology for Isolating Transgene-Free Mutants Generated by CRISPR. , 2017, Progress in molecular biology and translational science.

[31]  Hitoshi Sakakibara,et al.  An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice , 2019, Nature Plants.

[32]  K. Edwards,et al.  A simple method for spray-on gene editing in planta , 2019, bioRxiv.

[33]  Yunde Zhao,et al.  Specific and heritable gene editing in Arabidopsis , 2014, Proceedings of the National Academy of Sciences.

[34]  J. Sanford.  Biolistic plant transformation , 1990 .

[35]  Detlef Weigel,et al.  A proposed regulatory framework for genome-edited crops , 2016, Nature Genetics.

[36]  Rongchen Wang,et al.  Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants. , 2018, Molecular plant.

[37]  S. Gelvin,et al.  Factors influencing Agrobacterium-mediated transient expression of gusA in rice , 1992, Plant Molecular Biology.

[38]  Yunde Zhao,et al.  Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants. , 2019, Methods in molecular biology.

[39]  X. Deng,et al.  Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene , 2016, Proceedings of the National Academy of Sciences.

[40]  H. Puchta,et al.  From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas , 2019, aBIOTECH.

[41]  M. Estelle,et al.  Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development , 2015, Proceedings of the National Academy of Sciences.

[42]  K. Musunuru,et al.  What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine? , 2017, Circulation. Cardiovascular genetics.

[43]  D. Luo,et al.  Cytoplasmic Male Sterility of Rice with Boro II Cytoplasm Is Caused by a Cytotoxic Peptide and Is Restored by Two Related PPR Motif Genes via Distinct Modes of mRNA Silencing[W] , 2006, The Plant Cell Online.

[44]  Lei S. Qi,et al.  When genome editing goes off-target , 2019, Science.

[45]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[46]  Yunde Zhao,et al.  An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing1[OPEN] , 2016, Plant Physiology.

[47]  Markita P Landry,et al.  Carbon nanotube–mediated DNA delivery without transgene integration in intact plants , 2019, Nature Protocols.

[48]  Cong Chen,et al.  Mitochondrial ORFH79 is Essential for Drought and Salt Tolerance in Rice. , 2015, Plant & cell physiology.

[49]  F. Gao,et al.  ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice. , 2013, The New phytologist.

[50]  Joshua K Young,et al.  Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes , 2016, Nature Communications.

[51]  D. Voytas,et al.  Non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases. , 2015, Molecular plant.

[52]  K. Jung,et al.  T-DNA insertional mutagenesis for functional genomics in rice. , 2000, The Plant journal : for cell and molecular biology.

[53]  L. Steinmetz,et al.  Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos , 2019, Science.

[54]  Yushuang Guo,et al.  Cas9‐PF, an early flowering and visual selection marker system, enhances the frequency of editing event occurrence and expedites the isolation of genome‐edited and transgene‐free plants , 2019, Plant biotechnology journal.

[55]  T. Spencer,et al.  Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA , 2009, Transgenic Research.

[56]  Renshan Zhu,et al.  The mechanism of ORFH79 suppression with the artificial restorer fertility gene Mt-GRP162. , 2013, The New phytologist.

[57]  Rui Zhang,et al.  Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers , 2017, Nature Plants.

[58]  F. Baluška,et al.  Nanosheets for Delivery of Biomolecules into Plant Cells. , 2017, Trends in Plant Science.

[59]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[60]  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.