Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion

Journal name:
Nature Biotechnology
Volume:
35,
Pages:
441–443
Year published:
DOI:
doi:10.1038/nbt.3833
Received
Accepted
Published online
Corrected online

Abstract

We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

At a glance

Figures

  1. Target-AID in rice.
    Figure 1: Target-AID in rice.

    (a) Schematic illustration for Target-AID. Cytidines located at positions −19 to −17 upstream of the PAM sequence on the top strand (the cytidine target range) were highly mutated. Nearby cytidines were subjected to mutagenesis at a lower frequency. (b) Selection for ALS mutants. IMZ-selected calli are shown (left). Scale bar, 1 cm. Right table indicates the frequency of IMZ resistance. (c) Sequence alignment of the ALS mutants induced by nCas9Os-PmCDA1At. (d) Regenerated mutant plants (left) and Sanger sequencing spectra of their ALS target region. Arrowheads indicate overlapping, heterogenic mutations. Scale bar, 5 cm. (e) ALS-assisted multiplex mutagenesis. Two targets (W483 and G590) in FTIP1e were combined with the ALS target (A96V) and selected for IMZ resistance. Mutagenesis at G590 was assessed by CAPS analysis. (f) Mutation frequency at ALS and FTIP1e targets. The transformed and IMZ-resistant calli were analyzed by CAPS at G590. CAPS-positive lines were sequenced to identify mutation types. For ALS, the common spontaneous resistance mutations (W548C/L and S627I) were also examined. X represents stop codon.

  2. Target-AID in tomato.
    Figure 2: Target-AID in tomato.

    (a) Target sequences in DELLA and ETR1. (b) Patterns of DNA modification observed in independent T0 lines. Plus (+), minus (−) and S represent DNA insertion, deletion and substitution, respectively. Numbers following symbols (+ or −) indicate length (in base pairs) of indels, whereas those following an S indicate the number of nucleotides substituted. (c) Representative substitution spectra and changes in amino acid residues in marker-free T1 plants. All figures show target sequences, PAM sequences and DNA modifications in bold red, bold black, and bold blue font, respectively. Line names (starting with #) correspond to those in Supplementary Figure 6.

  3. Application of Target-AID to rice by EGFP reporter assay and transgenic approach.
    Supplementary Fig. 1: Application of Target-AID to rice by EGFP reporter assay and transgenic approach.

    (a) Schematics of the switch-mEGFP reporter vector (top) and rice Target-AID (d/nCas9Os-PmCDA1At) vector (bottom). In the switch module (OFF), a stop codon is inserted immediately downstream of the initiation codon of the EGFP gene, as indicated at the top. LB/RB, left/right border of T-DNA. (b) Fluorescence microscopic images of the transformants of mEGFP marker and Target-AID. Upper panels (Bright field) show shape of calli. Lower panels show GFP signal observed by fluorescence microscopy. Shape of calli are outlined and superimposed. Bar = 1 mm. (c) Frequency of GFP positive calli for each transformant. (d) Sequence alignment of the edited reporter EGFP. The switch module sequence of pRIT3-mEGFP is indicated with the target sequence (red). Mutated positions are shown in blue. Amino acid translation is indicated at the top. (e) Electrophoretogram of CAPS analysis for Fig. 1e and f. 1054 bp PCR product consisting of 1034 bp genomic region and 20 bp primer sequence was digested by PvuII into two fragments (614 bp and 440 bp) in the presence of C-to-T mutation at G590.

  4. Deep sequencing analysis of ALS on- and off-target mutations induced by a series of Target-AID vectors in rice.
    Supplementary Fig. 2: Deep sequencing analysis of ALS on- and off-target mutations induced by a series of Target-AID vectors in rice.

    Target sequence is highlighted in grey and mismatched bases of the off-target are highlighted in light blue. Mutation frequency is shown and highlighted as indicated at the bottom right. Total indel frequency in the 60 bases (20 bases of target region plus upstream 10 bases and downstream 30 bases) and SNV frequency above 0.1% at each nucleotide position is shown and highlighted.

  5. Application of Target-AID to tomato.
    Supplementary Fig. 3: Application of Target-AID to tomato.

    (a) Engineered Cas9 nuclease expression vectors used for tomato study. Nucleases (Cas9At, nCas9At-PmCDA1Hs, and nCas9At-PmCDA1At) were under the control of the PcUbi promoter, and transcription was terminated by the Pea3A terminator from P. sativum. sgRNAs were under the control of the Arabidopsis U6 promoter (AtU6). T0 lines were selected on the basis of kanamycin resistance. For construction of ETR1site3-targeting vector, a backbone of nCas9At-PmCDA1At-2A was used. (b) Targeted mutagenesis frequencies in T0 and T1 tomato lines. ‘No. of T0 lines generated’, number of T0 lines generated for this study; ‘No. of T0 lines sequenced’, number of T0 lines subjected to sequencing analysis; ‘No. of genome-edited T0 lines’, number of transgenic lines with DNA modifications in T0 transgenic lines; ‘No. of T0 lines with indels and no. of lines with substitutions’, number of T0 lines harboring indels and/or substitutions, respectively; ‘No. of T1 lines sequenced’, number of independent T0 generation lines used for sequencing analysis in T1 lines; ‘No. of genome-edited T1 lines’, number of independent T0 generation plants with DNA modifications in T1 lines; ‘No. of T1 lines with indels and no. of T1 lines with substitutions’, number of independent T0 lines harboring indels and substitutions, respectively, in T1 lines. (c) Summary of mutation segregation patterns in T1 offspring plants. Numbers of plants T1 harboring Indel or substitution mutations with each genotype are shown. Genotypes are classified into nine categories including T1 plants with homozygous (Indel/Indel), heterozygous (Indel/WT) or biallelic (Indel/Indel') indels, those with homozygous (S/S), heterozygous (S/WT) or biallelic (S/S' and S/Indel) substitutions, those with WT genotype (WT/WT) and those that contain more than three different sequences (Chimera). Indel' and S' represent different patterns of indel and substitution from those of indel and S, respectively. 167 T1 plants possessed stable DNA substitution, except for T1 plants with chimera genotype.

  6. Target-AID vectors induce amino acid substitutions and phenotype in genome-edited tomato plants.
    Supplementary Fig. 4: Target-AID vectors induce amino acid substitutions and phenotype in genome-edited tomato plants.

    (a) Changes in amino acid residues in DELLA- or ETR1-targeted T1 plants. Substitutions in amino acid residues were due to corresponding DNA substitutions in T1 plants. Line names (starting with #) correspond to those in Supplementary Figure 6b. The marker gene-free plants are highlighted. (b) Change in amino acid residues in DELLA-targeted T2 (#3_2_4) and T1 (#27_9) plants. (c) Leaflet appearance in DELLA-targeted T2 (#3_2_4) and T1 (#27_9) plants with homozygous 2-bp substitution and 12-bp deletion, respectively. Leaflets are highly serrated in WT (arrows), whereas those in procera mutant and the DNA edited plants were less serrated and looked smoother than those in WT. The procera is a loss-of-function allele of DELLA gene which shows reduced leaf serration and was shown as a control to compare leaf phenotype. Bar = 10 mm.

  7. Deep sequencing analysis of on- and off-target sites induced by a series of Target-AID and Cas9 vectors in tomato.
    Supplementary Fig. 5: Deep sequencing analysis of on- and off-target sites induced by a series of Target-AID and Cas9 vectors in tomato.

    (a) Prediction of DELLA off-target sites in tomato. Potential off-target genomic regions were selected by CCTop (http://crispr.cos.uni-heidelberg.de/index.html), and top one off-target site was highlighted in light blue. Distance to the closest exon: 0 indicates that the target site and exon coordinate overlap; NA indicates that the target sites are farther than 100 kb from the next exon. Location of the target site: E, I, and - indicate exonic, intronic, and intergenic, respectively. (b) DELLA on- and off-target mutation frequencies induced by a series of Target-AID and Cas9 in tomato. Target sequence is highlighted in grey and mismatched bases of the off-target are in light blue. Mutation frequency above 0.1% is shown and highlighted as indicated at the bottom right. Total indel frequency in the 42 bases (20 bases of target region plus upstream 10 bases and downstream 12 bases) and SNV frequency above 0.1% at each nucleotide position is shown and highlighted.

  8. Marker gene-free DELLA- or ETR1-targeted transgenic plants with stably inherited DNA mutations.
    Supplementary Fig. 6: Marker gene-free DELLA- or ETR1-targeted transgenic plants with stably inherited DNA mutations.

    (a) Presence of the kanamycin-resistance marker gene (NPT II) was tested by PCR analysis in T1 plants harboring DNA modifications. PCR analysis was conducted to confirm the absence of the kanamycin-resistance marker gene (NPT II, upper panel) in the genome of progenies derived from T0 lines with engineered Cas9 vectors. Lower panel indicates PCR amplification of the endogenous ACTIN gene, as a positive control (PC) for genomic DNA extraction. The negative control (NC) represents no DNA template added in PCR reaction mixture to prove that no DNA was contaminated in the PCR reaction. # indicates parental line, whereas following numbers indicate individual offspring. Line #3BC1_6 is an F1 plant derived from a cross between line #3 (T0 line) and the WT. WT, wild-type tomato plants (negative control of PCR for NPT II amplification). (b) The mutation spectrum in marker-gene free plants that correspond to those examined in (a). Primary null-segregant, the first generation that carried no transgene; Zygosity, type of stably inherited mutation; Next generation, self-pollinated siblings of primary null-segregant; No. of plants examined, number of transgenic plants used for calculation of mutation efficiency or heritable frequency; Ratio of segregated mutation, percentage of mutation patterns found in the next generation; N.D., Not determined.

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Primary accessions

Sequence Read Archive

Change history

Corrected online 05 April 2017
In the version of this article initially published, in the first paragraph after the abstract, a reference for Komor et al. was left out. The reference is now inserted in line 5 as “the base-editing1,18 construct”, replacing “with a construct.” The error has been corrected for the print, PDF and HTML versions of this article.

References

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Author information

  1. These authors contributed equally to this work.

    • Zenpei Shimatani,
    • Sachiko Kashojiya &
    • Mariko Takayama

Affiliations

  1. Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.

    • Zenpei Shimatani,
    • Takayuki Arazoe,
    • Hisaki Ishii,
    • Hiroshi Teramura,
    • Keiji Nishida &
    • Akihiko Kondo
  2. Faculty of Life and Environmental Sciences, University of Tsukuba, Gene Research Center, Tsukuba, Japan.

    • Sachiko Kashojiya,
    • Mariko Takayama,
    • Tsuyoshi Yamamoto,
    • Hiroki Komatsu,
    • Kenji Miura,
    • Hiroshi Ezura &
    • Tohru Ariizumi
  3. Laboratory of Genetics and Breeding Science, Faculty of Agriculture, Meijo University, Nagoya, Japan.

    • Rie Terada
  4. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.

    • Akihiko Kondo

Contributions

Z.S., H.I., H.T. and R.T. performed rice experiments. M.T., S.K., T.Y. and H.K. performed tomato experiments. K.N. and T. Arazoe performed NGS analysis. K.N. designed and constructed plasmids. Z.S., T. Ariizumi, R.T. and K.N. designed the experiments. Z.S., K.N., M.T., S.K. and T. Ariizumi, wrote the manuscript. K.N., T. Ariizumi, K.M., H.E. and A.K. supervised the project.

Competing financial interests

K.N., Z.S., T. Arazoe and A.K. have filed a patent related to the rice study as PCT/JP2016/085075. S.K., M.T., T.Y., K.M., T. Ariizumi and H.E. have filed a patent related to the tomato study as JP 2017-019921.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Application of Target-AID to rice by EGFP reporter assay and transgenic approach. (229 KB)

    (a) Schematics of the switch-mEGFP reporter vector (top) and rice Target-AID (d/nCas9Os-PmCDA1At) vector (bottom). In the switch module (OFF), a stop codon is inserted immediately downstream of the initiation codon of the EGFP gene, as indicated at the top. LB/RB, left/right border of T-DNA. (b) Fluorescence microscopic images of the transformants of mEGFP marker and Target-AID. Upper panels (Bright field) show shape of calli. Lower panels show GFP signal observed by fluorescence microscopy. Shape of calli are outlined and superimposed. Bar = 1 mm. (c) Frequency of GFP positive calli for each transformant. (d) Sequence alignment of the edited reporter EGFP. The switch module sequence of pRIT3-mEGFP is indicated with the target sequence (red). Mutated positions are shown in blue. Amino acid translation is indicated at the top. (e) Electrophoretogram of CAPS analysis for Fig. 1e and f. 1054 bp PCR product consisting of 1034 bp genomic region and 20 bp primer sequence was digested by PvuII into two fragments (614 bp and 440 bp) in the presence of C-to-T mutation at G590.

  2. Supplementary Figure 2: Deep sequencing analysis of ALS on- and off-target mutations induced by a series of Target-AID vectors in rice. (118 KB)

    Target sequence is highlighted in grey and mismatched bases of the off-target are highlighted in light blue. Mutation frequency is shown and highlighted as indicated at the bottom right. Total indel frequency in the 60 bases (20 bases of target region plus upstream 10 bases and downstream 30 bases) and SNV frequency above 0.1% at each nucleotide position is shown and highlighted.

  3. Supplementary Figure 3: Application of Target-AID to tomato. (171 KB)

    (a) Engineered Cas9 nuclease expression vectors used for tomato study. Nucleases (Cas9At, nCas9At-PmCDA1Hs, and nCas9At-PmCDA1At) were under the control of the PcUbi promoter, and transcription was terminated by the Pea3A terminator from P. sativum. sgRNAs were under the control of the Arabidopsis U6 promoter (AtU6). T0 lines were selected on the basis of kanamycin resistance. For construction of ETR1site3-targeting vector, a backbone of nCas9At-PmCDA1At-2A was used. (b) Targeted mutagenesis frequencies in T0 and T1 tomato lines. ‘No. of T0 lines generated’, number of T0 lines generated for this study; ‘No. of T0 lines sequenced’, number of T0 lines subjected to sequencing analysis; ‘No. of genome-edited T0 lines’, number of transgenic lines with DNA modifications in T0 transgenic lines; ‘No. of T0 lines with indels and no. of lines with substitutions’, number of T0 lines harboring indels and/or substitutions, respectively; ‘No. of T1 lines sequenced’, number of independent T0 generation lines used for sequencing analysis in T1 lines; ‘No. of genome-edited T1 lines’, number of independent T0 generation plants with DNA modifications in T1 lines; ‘No. of T1 lines with indels and no. of T1 lines with substitutions’, number of independent T0 lines harboring indels and substitutions, respectively, in T1 lines. (c) Summary of mutation segregation patterns in T1 offspring plants. Numbers of plants T1 harboring Indel or substitution mutations with each genotype are shown. Genotypes are classified into nine categories including T1 plants with homozygous (Indel/Indel), heterozygous (Indel/WT) or biallelic (Indel/Indel') indels, those with homozygous (S/S), heterozygous (S/WT) or biallelic (S/S' and S/Indel) substitutions, those with WT genotype (WT/WT) and those that contain more than three different sequences (Chimera). Indel' and S' represent different patterns of indel and substitution from those of indel and S, respectively. 167 T1 plants possessed stable DNA substitution, except for T1 plants with chimera genotype.

  4. Supplementary Figure 4: Target-AID vectors induce amino acid substitutions and phenotype in genome-edited tomato plants. (198 KB)

    (a) Changes in amino acid residues in DELLA- or ETR1-targeted T1 plants. Substitutions in amino acid residues were due to corresponding DNA substitutions in T1 plants. Line names (starting with #) correspond to those in Supplementary Figure 6b. The marker gene-free plants are highlighted. (b) Change in amino acid residues in DELLA-targeted T2 (#3_2_4) and T1 (#27_9) plants. (c) Leaflet appearance in DELLA-targeted T2 (#3_2_4) and T1 (#27_9) plants with homozygous 2-bp substitution and 12-bp deletion, respectively. Leaflets are highly serrated in WT (arrows), whereas those in procera mutant and the DNA edited plants were less serrated and looked smoother than those in WT. The procera is a loss-of-function allele of DELLA gene which shows reduced leaf serration and was shown as a control to compare leaf phenotype. Bar = 10 mm.

  5. Supplementary Figure 5: Deep sequencing analysis of on- and off-target sites induced by a series of Target-AID and Cas9 vectors in tomato. (107 KB)

    (a) Prediction of DELLA off-target sites in tomato. Potential off-target genomic regions were selected by CCTop (http://crispr.cos.uni-heidelberg.de/index.html), and top one off-target site was highlighted in light blue. Distance to the closest exon: 0 indicates that the target site and exon coordinate overlap; NA indicates that the target sites are farther than 100 kb from the next exon. Location of the target site: E, I, and - indicate exonic, intronic, and intergenic, respectively. (b) DELLA on- and off-target mutation frequencies induced by a series of Target-AID and Cas9 in tomato. Target sequence is highlighted in grey and mismatched bases of the off-target are in light blue. Mutation frequency above 0.1% is shown and highlighted as indicated at the bottom right. Total indel frequency in the 42 bases (20 bases of target region plus upstream 10 bases and downstream 12 bases) and SNV frequency above 0.1% at each nucleotide position is shown and highlighted.

  6. Supplementary Figure 6: Marker gene-free DELLA- or ETR1-targeted transgenic plants with stably inherited DNA mutations. (256 KB)

    (a) Presence of the kanamycin-resistance marker gene (NPT II) was tested by PCR analysis in T1 plants harboring DNA modifications. PCR analysis was conducted to confirm the absence of the kanamycin-resistance marker gene (NPT II, upper panel) in the genome of progenies derived from T0 lines with engineered Cas9 vectors. Lower panel indicates PCR amplification of the endogenous ACTIN gene, as a positive control (PC) for genomic DNA extraction. The negative control (NC) represents no DNA template added in PCR reaction mixture to prove that no DNA was contaminated in the PCR reaction. # indicates parental line, whereas following numbers indicate individual offspring. Line #3BC1_6 is an F1 plant derived from a cross between line #3 (T0 line) and the WT. WT, wild-type tomato plants (negative control of PCR for NPT II amplification). (b) The mutation spectrum in marker-gene free plants that correspond to those examined in (a). Primary null-segregant, the first generation that carried no transgene; Zygosity, type of stably inherited mutation; Next generation, self-pollinated siblings of primary null-segregant; No. of plants examined, number of transgenic plants used for calculation of mutation efficiency or heritable frequency; Ratio of segregated mutation, percentage of mutation patterns found in the next generation; N.D., Not determined.

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