Effect of Histone Deacetylase Inhibitor（HDACi）on CRISPR Editing Efficiency of Wheat and Transcriptomics Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2020-0645

Abstract

Abstract:

This study focuses on the relationship between histone deacetylase inhibitors（HDACi）and chromatin status and CRISPR/Cas9 editing efficiency. Different concentrations of nicotinamide（0,2.5,and 5 mmol/L）and sodium butyrate（0,5,and 10 mmol/L）were used to treat wheat seedlings for 7 d and 14 d,and the results showed that sodium butyrate seriously inhibited wheat growth,however,nicotinamide had little effect on it. Moreover,transcriptome sequencing of wheat seedlings treated with nicotinamide revealed some genes which were conducive to promoting the opening of the chromatin state,including 6 methyltransferase synthesis pathway genes. In addition,the TaAGO4a transgenic wheat materials in T₂ generation that had not been edited were treated with nicotinamide,and results showed that a heterozygous mutant plant with mutations in the homologous alleles on 3A and 3B chromosomes was detected in the 5 mmol/L treatment for 14 d,and the editing efficiency increased from 0 to 8.3%;there was no mutant found in other treatments or control group. This study proves that nicotinamide can indeed improve the editing efficiency of wheat genes as a new strategy.

Key words: wheat, histone deacetylation inhibitor, chromatin state, CRISPR/Cas9, RNA-seq

DAI Wen-shuang, LIU Hui-yun, DU Qing-guo, ZOU Cheng, WANG Ke. Effect of Histone Deacetylase Inhibitor（HDACi）on CRISPR Editing Efficiency of Wheat and Transcriptomics Analysis[J]. Biotechnology Bulletin, 2021, 37(1): 2-14.

Figures/Tables 12

References 65

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引物名称	引物序列（5'-3'）
AGO4a486F	AGACTTTGCCTATGATGGTGA
AGO4a1404R	AAATCTTACCTTAGCCCAATCA
Ago4aA1629R	ACTTACATACAACAAATGGCGATATA
Ago4aB749F	GAAACGACAGTCCAGGAAATG
Ago4aD698F	GTTGCTCATGTAACTCTGTTGTTG

引物名称	引物序列（5'-3'）
AGO4a486F	AGACTTTGCCTATGATGGTGA
AGO4a1404R	AAATCTTACCTTAGCCCAATCA
Ago4aA1629R	ACTTACATACAACAAATGGCGATATA
Ago4aB749F	GAAACGACAGTCCAGGAAATG
Ago4aD698F	GTTGCTCATGTAACTCTGTTGTTG

样本	非冗余序列数	Q20/%	Q30/%	GC/%	比对率/%
ctrl7d_1	59882027	97.08	93.21	56.72	91.21
ctrl7d_2	46792049	97.12	93.43	56.84	91.71
t7d-2.5_1	51954951	96.74	92.69	57.80	90.85
t7d-2.5_2	46624524	96.50	92.18	57.68	90.57
t7d-5_1	43606982	96.87	92.91	57.30	91.48
t7d-5_2	44386513	97.08	93.08	57.24	91.89
ctrl14d _1	47377531	97.23	93.32	57.09	91.90
ctrl14d _2	62520474	97.15	93.33	58.19	92.52
t14d-2.5_1	44413018	96.83	92.58	57.21	91.76
t14d-2.5_2	65941651	96.97	92.94	57.65	91.87
t14d-5_1	48123569	96.92	93.16	58.24	90.87
t14d-5_2	50461326	97.23	93.39	58.17	92.49

样本	非冗余序列数	Q20/%	Q30/%	GC/%	比对率/%
ctrl7d_1	59882027	97.08	93.21	56.72	91.21
ctrl7d_2	46792049	97.12	93.43	56.84	91.71
t7d-2.5_1	51954951	96.74	92.69	57.80	90.85
t7d-2.5_2	46624524	96.50	92.18	57.68	90.57
t7d-5_1	43606982	96.87	92.91	57.30	91.48
t7d-5_2	44386513	97.08	93.08	57.24	91.89
ctrl14d _1	47377531	97.23	93.32	57.09	91.90
ctrl14d _2	62520474	97.15	93.33	58.19	92.52
t14d-2.5_1	44413018	96.83	92.58	57.21	91.76
t14d-2.5_2	65941651	96.97	92.94	57.65	91.87
t14d-5_1	48123569	96.92	93.16	58.24	90.87
t14d-5_2	50461326	97.23	93.39	58.17	92.49

处理时间/d	GO terms	基因数目	类别	调控方式
7	nicotianamine synthase activity	15	MF	down
7	nicotianamine biosynthetic process	15	BP	down
7	O-methyltransferase activity	6	MF	down
7	transaminase activity	6	MF	up
7	cell division	4	BP	up