Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants

doi:10.16420/j.issn.0513-353x.2022-1168

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (12): 2591-2600.doi: 10.16420/j.issn.0513-353x.2022-1168

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants

XIONG Wenwen¹, LIU Huimin¹, CHEN Kaiyuan², DU Juan¹, SONG Botao¹^,^*(), JING Shenglin¹^,^*()

¹ Key Laboratory of Horticultural Plant Biology Ministry of Education，Key Laboratory of Potato Biology and Biotechnology，Ministry of Agriculture and Rural Affairs，School of Horticulture and Forestry，Huazhong Agricultural University，Wuhan 430070，China
² National Key Laboratory of Crop Genetic Improvement，College of Plant Science & Technology of Huazhong Agricultural University，Wuhan 430070，China

Received:2023-08-07 Revised:2023-10-17 Online:2023-12-25 Published:2023-12-29
Contact: SONG Botao, JING Shenglin

Abstract

Abstract:

The development of endogenous promoters driven CRISPR/Cas9 gene editing system in diploid potato cultivar AC142 and tetraploid cultivar E3 were reported. The StPDS gene was targeted by Agrobacterium-mediated potato microtuber transformation. Through the analysis of transformation efficiency and phenotypic mutation rate，HM4 driven by potato endogenous promoter StU6 and StUBI10 was selected as the optimal CRISPR vector. The short-term cycle of eight heat treatment（37 ℃ 24 h，22 ℃ 24 h）was performed on the lateral buds of 37 AC142 all-green lines and chimeric lines and four E3 all-green lines obtained from HM4 for 15 days. Phenotypic analysis and sequencing showed that short-term heat treatment of knockout lines could significantly improve the editing efficiency of CRISPR/Cas9. At room temperature，the phenotypic mutation rates of AC142 and E3 knockout lines were 16.2%（6/37）and 0（0/4），respectively. After one short-term heat treatment，the phenotypic mutation rates were increased to 54.1%（20/37）and 75%（3/4），respectively. Among them，AC142 knockout lines can obtain 10.8%（4/37） lines that maintain the albino phenotype，while E3 can only obtain 75%（3/4）chimeras with white spots. Four consecutive short-term heat treatments could result in 2.1%（3/144）albino mutants at room temperature in E3 knockout lines. TA cloning was performed on the albino lines after short-term heat treatment. The sequencing results showed that 8-12 single clones of the diploid knockout line CR-AC142-StPDS-17，21 and the tetraploid knockout line CR-E3-StPDS-3，four did not contain wild type，indicating that the knockout system in this study could induce mutations in the target sequence. The results provide a technical basis for functional genomics research and precise improvement of target traits in potato.

Key words: potatoes, gene editing, CRISPR/Cas9, StPDS, heat treatment

XIONG Wenwen, LIU Huimin, CHEN Kaiyuan, DU Juan, SONG Botao, JING Shenglin. Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants[J]. Acta Horticulturae Sinica, 2023, 50(12): 2591-2600.

Figures/Tables 9

Table 1 Primer sequence

引物名称 Primer name	引物序列（5′-3′） Sequence of primers
pJCV55-F	AGCTCTCCCATATGGTCGACAAGGAATCTTTAAACATACG
pJCV55-R	CGGGAATTGGATCCGGTACCTCTAGATATCTCGAGTGCGG
pJCV-StUBI10-F	AGCTCTCCCATATGGTCGACCCCACCTTGAATGGATATGC
pJCV-StUBI10-Bsp-R	GCCTGCTTTTTTGTACACTTGAAAGAGAAATTTGAAGGC
GTR-StPDS-F	CGGGTCTCCTGCACAATCTTCTGGTCGTGGCATGTTTTAGAGCTAGAA
GTR-StPDS-R	TAGGTCTCTAAACAACCGATACTACTGGAGGCATGCACCAGCCGGGAA
Cas9-Check-F	GAGATACGACGAGCACCACC
Cas9-Check-R	CTTCACGGTCACTTTCCGGT
PDS-Check-F	TTGAGAGTCCAAGGTAGTTCAGCTT
PDS-Check-R	GTTTACGTAGCATCTCACAGAAAAGAC

Fig. 1 gRNA design of two target sites of potato StPDS gene（A）and structure of CRISPR vector（B） HM1，HM2，HM3 and HM4 are carrier names，pRGEB32 is the carrier skeleton of HM1，and pJCV55 is the carrier skeleton of HM2，HM3 and HM4.

Table 2 Statistics of CRISPR vector transformation efficiency and phenotypic mutation rate of potato AC142 and E3

载体名称 Vector name	转化受体 Transforma- tion receptor	外植体总数 Total number of explants	抗性芽总数 Total number of resistant buds	二次生根数 Number of secondary roots	转化效率/% Transforma- tion efficiency	表型Phenotype				表型突变频率/% Phenotypic mutation rate
载体名称 Vector name	转化受体 Transforma- tion receptor	外植体总数 Total number of explants	抗性芽总数 Total number of resistant buds	二次生根数 Number of secondary roots	转化效率/% Transforma- tion efficiency	白化 Albino	嵌合 Mosaic	全绿 No albino		表型突变频率/% Phenotypic mutation rate
HM1	E3	106	105	1	1	0	0		1	0
HM1	AC142	164	103	0	0	0	0		0	0
HM2	E3	149	159	2	1	0	0		2	0
HM2	AC142	216	188	2	1	0	0		2	0
HM3	E3	147	157	2	1	0	0		2	0
HM3	AC142	581	521	41	8	2	2		37	10
HM4	E3	157	206	4	2	0	0		4	0
HM4	AC142	299	461	44	10	4	6		34	23

Fig. 2 Phenotypic diagram of potato AC142 and E3 wild type（WT）and its CRISPR knockout StPDS lines（CR）

Fig. 3 The heat treatment albino map of CRISPR knockout StPDS lines（CR）of potato AC142 and E3 A-B：the white green chimeric and albino lines obtained by knocking out StPDS in AC142;C-E：chimeric lines，axillary green returning lines，and albino lines obtained by knocking out StPDS in E3. HT→RT refers to the growth of albino buds at room temperature after one short-term heat treatments，2HT→RT and 4HT → RT refers to the growth of albino buds at room temperature after two and four consecutive short-term heat treatments. The red arrow refers to the albino bud after high temperature，which is taken down separately and grown at room temperature to observe whether it turns green.

Table 3 Effect of short-term heat treatment on the phenotype of CRISPR knockout StPDS lines of potato AC142 and E3

马铃薯 Potato	阳性苗株数 Number of transgenic lines	处理 Treatment	表型Phenotype			突变频率 Frequency of induced mutants
马铃薯 Potato	阳性苗株数 Number of transgenic lines	处理 Treatment	白化 Albino	嵌合 Mosaic	全绿 No albino	突变频率 Frequency of induced mutants
AC142	37	22 ℃常温Room temperature	0	6	31	6/37
AC142	37	37 ℃热处理Heat treatment	4	16	17	20/37
E3	4	22 ℃常温Room temperature	0	0	4	0/4
E3	4	37 ℃热处理Heat treatment	0	3	1	3/4

Fig. 4 Mutation types of target sites in gene editing lines RT：Room temperature treatment;HT：Heat treatment. The black bold font indicates the protospacer-adjacent motif sequences，the blue font represents the gRNA of the target sequence，and the red font is the point mutation site. The insertion and deletion are represented by + and-，respectively. The numbers in the left bracket of the target sequence represent the number of monoclonals for the corresponding treatment and mutation types，respectively. The number of bases in the brackets on the right represents the total number of bases inserted and deleted in the clone.

Table 4 Mutation types of target sites in gene editing lines

温度/℃ Temperature	突变类型 Indel types	碱基数/bp Number of base	位点1（%）gRNA 1 sites（%）	位点2（%）gRNA 2 sites（%）
22	点突变，单碱基缺失或插入Point mutation，single base deletion or insertion	1	5（12.5）	8（20.0）
	小片段缺失Small fragment deletion	3 ~ 31	5（12.5）	11（27.5）
	大片段缺失或插入Long fragment deletion or insertion	—	—	—
37	点突变，单碱基缺失或插入Point mutation，single base deletion or insertion	1	17（35.4）	12（25.0）
	小片段缺失Small fragment deletion	2 ~ 59	15（31.3）	26（54.2）
	大片段缺失或插入Long fragment deletion or insertion	73 ~ 1434	7（14.6）	7（14.6）

Fig. 5 TA clone PCR detection results of CR-E3-StPDS-4 knockout lines WT is the E3 wild type control，1-19 refer to 19 monoclonals of the target sequence of the CR-E3-StPDS-4 knockout line.

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Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants

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