Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

doi:10.1016/j.rsci.2018.11.001

Abstract

Abstract:

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Key words: genome editing, suicide gene, transgene killer CRISPR, Cas9, transgene-free

Yubing He, Min Zhu, Lihao Wang, Junhua Wu, Qiaoyan Wang, Rongchen Wang, Yunde Zhao. Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants[J]. Rice Science, 2019, 26(2): 109-117.

Figures/Tables 3

References 56

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T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#11	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	BI, -10/-C	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	70.00% (7/10)
	BI, -10/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	70.00% (7/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
#16	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	75.00% (6/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	25.00% (2/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	25.00% (2/8)
#24	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	43.75% (7/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	43.75% (7/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAG-----GCAGGCGCCGCGGG	6.25% (1/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	6.25% (1/16)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#29	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	60.00% (9/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	60.00% (9/15)
#32	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	53.85% (7/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	53.85% (7/13)
#36	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	14.29% (1/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
#42	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	75.00% (6/8)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#59	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	50.00% (7/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	50.00% (7/14)
WT, Wild-type Zhonghua 11 plant; HO, Homozygous mutation; HE, Heterozygous mutation; BI, Bi-allelic mutation. The underlined ‘AGG’ refers to the protospacer adjacent motif site required for Cas9 cleavage. ‘-’ refers to one base pair deletion. Lowercase letter ‘t’ in red refers to an insertion of a ‘T’. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#1	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (7/7)
#11	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	10.00% (1/10)
	BI, -10/-C	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	70.00% (7/10)
	BI, -10/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	70.00% (7/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
	HO, -10/-10	TCGAGTCGCGCCCGG----------GGCGCCGCGGG	20.00% (2/10)
#16	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	75.00% (6/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	25.00% (2/8)
	HE, WT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	25.00% (2/8)
#24	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	HO, +T/+T	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	25.00% (4/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	43.75% (7/16)
	BI, +T/-C	TCGAGTCGCGCCCGGAGTACCtTGCAGGCGCCGCGGG	43.75% (7/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAG-----GCAGGCGCCGCGGG	6.25% (1/16)
	BI, -TACCT/-C	TCGAGTCGCGCCCGGAGTA-CTGCAGGCGCCGCGGG	6.25% (1/16)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#27	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (18/18)
#29	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	13.33% (2/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	26.67% (4/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	60.00% (9/15)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	60.00% (9/15)
#32	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	HO, -ACCT/-ACCT	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	46.15% (6/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	53.85% (7/13)
	BI, -ACCT/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	53.85% (7/13)
#36	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	14.29% (1/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	57.14% (4/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	14.29% (1/7)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	14.29% (1/7)
#42	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	25.00% (2/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGT----GCAGGCGCCGCGGG	75.00% (6/8)
	BI, -ACCT/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	75.00% (6/8)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#52	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (10/10)
#59	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	42.86% (6/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	7.14% (1/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	50.00% (7/14)
	BI, -C/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	50.00% (7/14)
WT, Wild-type Zhonghua 11 plant; HO, Homozygous mutation; HE, Heterozygous mutation; BI, Bi-allelic mutation. The underlined ‘AGG’ refers to the protospacer adjacent motif site required for Cas9 cleavage. ‘-’ refers to one base pair deletion. Lowercase letter ‘t’ in red refers to an insertion of a ‘T’. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#5	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	50.00% (9/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	50.00% (9/18)
#17	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	33.33% (3/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
#22	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	41.18% (7/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	41.18% (7/17)
Protospacer adjacent motif site AGG is underlined. Zhonghua 11 plants were used as wild-type (WT). ‘+’ refers to base pair insertion. ‘-’ refers to base pair deletion. Lowercase letter ‘c’ and ‘g’ in red refer to insertions of ‘C’ and ‘G’, respectively. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.

T₀ plant	Genotype of T₁ plant	Sequence analysis	Segregation
WT	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#3	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	100.00% (22/22)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#4	HO, -CC/-CC	TCGAGTCGCGCCCGGAGTA--TGCAGGCGCCGCGGG	100.00% (9/9)
#5	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	16.67% (3/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	HO, -CCT/-CCT	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	33.33% (6/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTA---GCAGGCGCCGCGGG	50.00% (9/18)
	BI, -CCT/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	50.00% (9/18)
#17	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, -C/-C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	HO, +C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	11.11% (1/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTAC-TGCAGGCGCCGCGGG	33.33% (3/9)
	BI, -C/+C	TCGAGTCGCGCCCGGAGTACCcTGCAGGCGCCGCGGG	33.33% (3/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
	WT	TCGAGTCGCGCCCGGAGTACCTGCAGGCGCCGCGGG	22.22% (2/9)
#22	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, -T/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	35.29% (6/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	HO, +G/+G	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	23.53% (4/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACCgTGCAGGCGCCGCGGG	41.18% (7/17)
	BI, +G/-T	TCGAGTCGCGCCCGGAGTACC-GCAGGCGCCGCGGG	41.18% (7/17)
Protospacer adjacent motif site AGG is underlined. Zhonghua 11 plants were used as wild-type (WT). ‘+’ refers to base pair insertion. ‘-’ refers to base pair deletion. Lowercase letter ‘c’ and ‘g’ in red refer to insertions of ‘C’ and ‘G’, respectively. Number of plants for the genotype and the total number of the T₁ plants from each T₀ plant analyzed are shown in the parenthesis.