PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

[1]  Guoying Wang,et al.  Generation of Transgene-Free Semidwarf Maize Plants by Gene Editing of Gibberellin-Oxidase20-3 Using CRISPR/Cas9 , 2020, Frontiers in Plant Science.

[2]  A. Alok,et al.  The present and potential future methods for delivering CRISPR/Cas9 components in plants , 2020, Journal of Genetic Engineering and Biotechnology.

[3]  Yuriko Osakabe,et al.  Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering , 2020, BMC Plant Biology.

[4]  W. Gordon-Kamm,et al.  Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template , 2020, bioRxiv.

[5]  Jeffry D. Sander,et al.  Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion , 2020, Frontiers in Plant Science.

[6]  Jianbing Yan,et al.  Genetic variants and underlying mechanisms influencing variance heterogeneity in maize. , 2020, The Plant journal : for cell and molecular biology.

[7]  Joshua K Young,et al.  Superior field performance of waxy corn engineered using CRISPR–Cas9 , 2020, Nature Biotechnology.

[8]  Jianbing Yan,et al.  High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize[OPEN] , 2020, Plant Cell.

[9]  B. Je,et al.  The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses , 2019, Proceedings of the National Academy of Sciences.

[10]  Nicholas A. Rossi,et al.  Inference of CRISPR Edits from Sanger Trace Data , 2019, bioRxiv.

[11]  Sunghwa Choe,et al.  DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants , 2019, Transgenic Research.

[12]  L. Mao,et al.  Supersweet and waxy: meeting the diverse demands for specialty maize by genome editing , 2019, Plant biotechnology journal.

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

[14]  Shujie Dong,et al.  One-step genome editing of elite crop germplasm during haploid induction , 2019, Nature Biotechnology.

[15]  G. Ingram,et al.  Single and multiple gene knockouts by CRISPR–Cas9 in maize , 2019, Plant Cell Reports.

[16]  T. Sprink,et al.  DNA-Free Genome Editing: Past, Present and Future , 2019, Front. Plant Sci..

[17]  Martin J. Aryee,et al.  Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize , 2018, Plant biotechnology journal.

[18]  Michael J. Bernstein,et al.  Revisiting Risk Governance of GM Plants: The Need to Consider New and Emerging Gene-Editing Techniques , 2018, Front. Plant Sci..

[19]  P. Hofvander,et al.  Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. , 2018, Physiologia plantarum.

[20]  B. Bohanec,et al.  DNA-Free Genome Editing of Brassica oleracea and B. rapa Protoplasts Using CRISPR-Cas9 Ribonucleoprotein Complexes , 2018, Front. Plant Sci..

[21]  Yan Liu,et al.  Generation of Transgene-Free Maize Male Sterile Lines Using the CRISPR/Cas9 System , 2018, Front. Plant Sci..

[22]  Yunhan Hong,et al.  Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells , 2018, Biology Open.

[23]  Jing Zhang,et al.  High‐efficiency genome editing using a dmc1 promoter‐controlled CRISPR/Cas9 system in maize , 2018, Plant biotechnology journal.

[24]  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.

[25]  A. Pattanayak,et al.  Insights into maize genome editing via CRISPR/Cas9 , 2018, Physiology and Molecular Biology of Plants.

[26]  B. Reiss,et al.  Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9‐induced DNA double‐stranded breaks in Arabidopsis thaliana , 2017, The Plant journal : for cell and molecular biology.

[27]  C. Jung,et al.  CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus)1 , 2017, Plant Physiology.

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

[29]  V. Walbot,et al.  An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize , 2016, Plant biotechnology journal.

[30]  Hongyu Wang,et al.  ARGOS8 variants generated by CRISPR‐Cas9 improve maize grain yield under field drought stress conditions , 2016, Plant biotechnology journal.

[31]  R. Viola,et al.  DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins , 2016, Front. Plant Sci..

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

[33]  Rainer Fischer,et al.  Patterns of CRISPR/Cas9 activity in plants, animals and microbes , 2016, Plant biotechnology journal.

[34]  R. Barrangou,et al.  Applications of CRISPR technologies in research and beyond , 2016, Nature Biotechnology.

[35]  Wei Zhang,et al.  High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize , 2016, BMC Biotechnology.

[36]  Y. Zong,et al.  An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons. , 2016, Current protocols in plant biology.

[37]  C. N. Kanchiswamy.  DNA-free genome editing methods for targeted crop improvement , 2016, Plant Cell Reports.

[38]  Jin-Soo Kim,et al.  Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins , 2016, Plant Cell Reports.

[39]  Jinsheng Lai,et al.  Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9. , 2016, Journal of genetics and genomics = Yi chuan xue bao.

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

[41]  Joshua K Young,et al.  Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN] , 2015, Plant Physiology.

[42]  Rainer Fischer,et al.  The CRISPR/Cas9 system for plant genome editing and beyond. , 2015, Biotechnology advances.

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

[44]  Xingxu Huang,et al.  sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites , 2014, PloS one.

[45]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[46]  Kang Zhang,et al.  Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. , 2014, Journal of genetics and genomics = Yi chuan xue bao.

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

[48]  A. Grobman.  Maize: Origin, Domestication, and Evolution of Maize , 2013 .

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

[50]  J. Sheen.  Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. , 1991, The Plant cell.

[51]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.