CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside

doi:10.13560/j.cnki.biotech.bull.1985.2024-0520

Abstract

Abstract:

【Objective】 Because cassava（Manihot esculenta Crantz）contains potentially toxic cyanogenic glucosides, its food safety is affected, which leads to the increase of processing costs. It is of great significance to cultivate cassava with low cyanogenic glycosides by biotechnology.【Method】 CRISPR/Cas9 technology was used to edit the α-hydroxynitrile lyase gene MeHNL. This gene encodes α-hydroxynitrile lyase that catalyzes the decomposition of cyanogenic glycosides. The editing target is located on its first exon, and 27 positive plants were obtained through Agrobacterium-mediated stable transformation.【Result】Sequencing analysis indicated successful editing in 26 out of 27 transgenic plants, and a high editing efficiency was achieved by 96.3%. The editing types predominantly comprised base insertions and deletions, alongside minor base substitutions and large fragment deletions. HCN colorimetric kit and HPLC analysis confirmed a significant reduction in hydrocyanic acid and cyanogenic glycosides in the mutant lines. Additionally, transgenic plants demonstrated slender leaves compared to non-transgenic counterparts, implicating MeHNL's impact on plant growth and development.【Conclusion】The cassava germplasm with low cyanide is obtained by CRISPR/Cas9 technology, which provides materials for exploring the study of cyanogenic glycoside metabolism on cassava growth and development.

Key words: cassava, gene editing, HNL, low cyanide, cyanogenic glycosides

TONG Wei-jing, LUO Shu, LU Xin-lu, SHEN Jian-fu, LU Bai-yi, LI Kai-mian, MA Qiu-xiang, ZHANG Peng. CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside[J]. Biotechnology Bulletin, 2024, 40(9): 11-19.

Figures/Tables 7

References 35

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引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Use
target-Fp	GATTGCTTGGAGAAACTCCCTCAAG	Target gene
target-Rp	AAACCTTGAGGGAGTTTCTCCAAGC	Target gene
M13-Fp	TGTAAAACGACGGCCAGT	Identification of positive clones
Hpt II-Fp	TTCTACACAGCCATCGGTCC	Identification of positive transgenic plants
Hpt II-Rp	CCCATGTGTATCACTGGCAA	Identification of positive transgenic plants
HNLgRNA-Fp	GCACCACTCACAGAAAAATCCAAAG	PCR amplification of target region
HNLgRNA-Rp	ATGTGGCTTAATTAGAATACCGTCT	PCR amplification of target region

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Use
target-Fp	GATTGCTTGGAGAAACTCCCTCAAG	Target gene
target-Rp	AAACCTTGAGGGAGTTTCTCCAAGC	Target gene
M13-Fp	TGTAAAACGACGGCCAGT	Identification of positive clones
Hpt II-Fp	TTCTACACAGCCATCGGTCC	Identification of positive transgenic plants
Hpt II-Rp	CCCATGTGTATCACTGGCAA	Identification of positive transgenic plants
HNLgRNA-Fp	GCACCACTCACAGAAAAATCCAAAG	PCR amplification of target region
HNLgRNA-Rp	ATGTGGCTTAATTAGAATACCGTCT	PCR amplification of target region

Type	MeHNL-sgRNA target sites	Indel	Lines
WT	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA		WT
1	CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA	-16	L1
2	CTTGGAGAAACTCCCTCAAGGGGAAAAAGGTCATCA CTTGGAGAAACTCCC - -AAGGAGAAGAGGTCATCA	+1, -2, 2	L2, L7
3	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+1, +3	L3, L11
4	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA	+1	L4
5	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACT- - - - - - - - - - - - - - - - - - - - GTCATCA	+1, -16	L5
6	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+3	L6, L9, L16
7	CTTGGAGAAACTCCCTCGAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC - - - AGGGGAAAAGGTCATCA	+1, -3	L10
8	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCCAAAGGGGAAAAGGTCATCA	+1, +2	L12, L18, L31
9	CTTGGAGAAACTCCCTC - - - - - - - - - - - - - - - - CATCA CTTGGAGAAACTCCCGTCAAGGGGAAAAGGTCATCA	-13, +1	L13
10	CTTGGAGAAACTCC- - - - - - -GGCAAAAGGTCATCA CTTGGAGAAACTAGCTCCAAGGGGAAAAGGTCATCA	-7, +1, +1, 2	L14
11	CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA	-1	L15
12	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC- -AATAAGGGGAAAAGGTCATCA	-2, +3	L17
13	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCA CTTGGAGAAACTCCC- -AAAAGGGGAAAAGGTCATCA	+1, -2, +2	L19, L32
14	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCT- -TCA AAGGGGAAAAGGTCATCA	+1, -2, +3	L21
15	CTTGGAGAAACTCCCTCAAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTGGAAGGGGAAAAGGTCATCA	+1, +3	L24
16	CTCTGAACCCTTATTGACTTTCTTGGAGAAACTCCCTCAAGGGG CTCTGAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGGGG	-31	L25
17	CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA	-8	L26
18	CTTGGAGAAACTCCCTCAAG - - - - - - - - - - - - - - - - - - TCATCA CTTGGAGAAA - - - - - - GGGGAAATGAAGGGGAAAAGGTCATCA	-9, -7, +9	L29

Type	MeHNL-sgRNA target sites	Indel	Lines
WT	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA		WT
1	CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA CTTGG - - - - - - - - - - - - - - - -- - - GGAAAAGGTCATCA	-16	L1
2	CTTGGAGAAACTCCCTCAAGGGGAAAAAGGTCATCA CTTGGAGAAACTCCC - -AAGGAGAAGAGGTCATCA	+1, -2, 2	L2, L7
3	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+1, +3	L3, L11
4	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA	+1	L4
5	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACT- - - - - - - - - - - - - - - - - - - - GTCATCA	+1, -16	L5
6	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCAAAAAGGGGAAAAGGTCATCA	+3	L6, L9, L16
7	CTTGGAGAAACTCCCTCGAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC - - - AGGGGAAAAGGTCATCA	+1, -3	L10
8	CTTGGAGAAACTCCCTCTAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCCAAAGGGGAAAAGGTCATCA	+1, +2	L12, L18, L31
9	CTTGGAGAAACTCCCTC - - - - - - - - - - - - - - - - CATCA CTTGGAGAAACTCCCGTCAAGGGGAAAAGGTCATCA	-13, +1	L13
10	CTTGGAGAAACTCC- - - - - - -GGCAAAAGGTCATCA CTTGGAGAAACTAGCTCCAAGGGGAAAAGGTCATCA	-7, +1, +1, 2	L14
11	CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTC- AGGGGAAAAGGTCATCA	-1	L15
12	CTTGGAGAAACTCCCTCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCC- -AATAAGGGGAAAAGGTCATCA	-2, +3	L17
13	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCA CTTGGAGAAACTCCC- -AAAAGGGGAAAAGGTCATCA	+1, -2, +2	L19, L32
14	CTTGGAGAAACTCCCTCCAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCT- -TCA AAGGGGAAAAGGTCATCA	+1, -2, +3	L21
15	CTTGGAGAAACTCCCTCAAAGGGGAAAAGGTCATCA CTTGGAGAAACTCCCTCTGGAAGGGGAAAAGGTCATCA	+1, +3	L24
16	CTCTGAACCCTTATTGACTTTCTTGGAGAAACTCCCTCAAGGGG CTCTGAA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -AAGGGG	-31	L25
17	CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA CTTGGAGAAA - - - - - - - - AGGGGAAAAGGTCATCA	-8	L26
18	CTTGGAGAAACTCCCTCAAG - - - - - - - - - - - - - - - - - - TCATCA CTTGGAGAAA - - - - - - GGGGAAATGAAGGGGAAAAGGTCATCA	-9, -7, +9	L29