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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (10): 1522-1534.doi: 10.3724/SP.J.1006.2019.84130

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Enhancing CRISPR/Cas9 genomic editing efficiency based on optimization of sgRNA of Gossypium barbadense L.

LI Ji-Yang1,2,HU Yan1,YAO Rui2,DAI Pei-Hong1,*(),LIU Xiao-Dong1,*()   

  1. 1Agricultural College, Xinjiang Agricultural University / Key Laboratory of Agricultural Biotechnology, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
    2Life Sciences College, Shihezi University, Shihezi 832000, Xinjiang, China
  • Received:2018-10-16 Accepted:2019-05-12 Online:2019-10-12 Published:2019-09-10
  • Contact: Pei-Hong DAI,Xiao-Dong LIU E-mail:40836520@qq.com;xiaodongliu75@aliyun.com
  • Supported by:
    This study was supported by the Nanjing Agricultural University-Xinjiang Agricultural University Joint Fund Project(KYYJ201603);Xinjiang Agricultural University Crop Science Key Discipline Development Fund Project.

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Abstract:

The CRISPR/Cas9 genome editing system has been established in many crops. The advantages of its directional creation of mutants are increasingly favored by researchers. However, the CRISPR/Cas9 genome editing technology targets the editing of the target gene and also triggers off-target effects at different frequencies, which determine the reliability of the genome editing system. This study was based on the previous CRISPR/Cas9 genome editing system established in the island-cotton somatic cells. Edit the vector by constructing different codon optimization methods for Cas9, different numbers of PAM sites and different target sites, and the difference in editing efficiency and off-target effect were analyzed and compared. There was no significant difference in editing effects and off-target effects caused by Cas9-edited vectors with different optimal codons. The partially double-sgRNA had significantly higher editing efficiency and significantly lower off-target efficiency than the single sgRNA; the editing efficiency of the transformed No shift sgRNA target sequence was significantly higher than that of the Shift sgRNA and the former had a significant decrease in off-target efficiency relative to the latter. Therefore, using No shift sgRNA type target sequence can effectively improve the editing efficiency and significantly reduce the off-target efficiency, thus laying a theoretical foundation for optimizing the CRISPR/Cas9 mediated island cotton genome editing system and accurately and efficiently creating island cotton functional gene mutants in the future.

Key words: cotton, CRISPR/Cas9, editing efficiency, off-target efficiency, sgRNA type

Table 1

Primers used in this study"

序列名称
Sequence name		 序列
Sequence (5'-3')		 PAM位点序列
PAM site sequence
Shift GbU6-4PERA1-sg2F GATTGTCTTTCGCAGAATGCATGA CGG
Shift GbU6-4PERA1-sg2R AAACTCATGCATTCTGCGAAAGAC
GbU6-4PERA1-sg2F GATTGTTCGCAGAATGCATGACGG TGG
GbU6-4PERA1-sg2R AAACCCGTCATGCATTCTGCGAAC
GbU6-5PGGB-sg1F AAGTGTGTCGAAGTACTGAAGCGG CGG
GbU6-5PGGB-sg1R AAACCCGCTTCAGTACTTCGACAC
GbU6-4PGGB-sg2F GATTGTCTTTCAGTCTTATGATGG TGG
GbU6-4PGGB-sg2R AAACCCATCATAAGACTGAAAGAC
Shift GbU6-5PGGB-sg1F AAGTGCTCTGTCGAAGTACTGAAG CGG
Shift GbU6-5PGGB-sg1R AAACCTTCAGTACTTCGACAGAGC
Shift GbU6-4PGGB-sg2F GATTGTGTTCTTTCAGTCTTATGA TGG
Shift GbU6-4PGGB-sg2R AAACTCATAAGACTGAAAGAACAC
Test GGB-sg1F AAGTGGAAAGAGAATGGCGAC
Test GGB-sg1R AGCTAATTCGCTATTGCAATCAATC
Test GGB-sgR ACCATGTGATTCTAAACCAGG
Test ERA1-sg1F TAATAGGTGGTCAGGTTATGC
Test ERA1-sg1R AGGAGACTTGCAACCTGATC
FJ-1F GTAAAACGACGGCCAG
FJ-1R CAGGAAACAGCTATGAC
FJ-2F TAAACTGAAGGCGGGAAACG
FJ-2R CGGTTCTGTCAGTTCCAAACG

Fig. 1

No shift sgRNA and Shift sgRNA target sequence characteristics and mode of action The red box identifies the PAM site for the action of CRISPR/Cas9."

Fig. 2

Sketch map of genome editing structure of cotton (Gossypium barbadense L.) A: editing vector containing Cas9I; B: editing vector containing Cas9II."

Table 2

Information of off target sequence"

脱靶序列名称
Name for sequence of off target		 序列
Sequence (5°-3°)		 预测脱靶率
Predict the off target rate
OT-GGB-sgRNA1-1 GGTCCAAGGACTGAAGCTGTGG 0.311
OT-GGB-sgRNA1-2 GGTTGAAGTAGTAAAGCGGAGG 0.177
OT-Shift GGB-sgRNA1-1 TCCAGTCGAAATACTGAAGAGG 0.408
OT-Shift GGB-sgRNA1-2 CTCTGTTGATGAACTGATGGGG 0.287

Table 3

Information of off target sequence primer"

序列名称
Sequence name		 序列
Sequence (5°-3°)
OTtest-G1-1F CCTTGAAAGTTGCTTCCGAC
OTtest-G1-1R GAAACTGCCTTTTTTGACACC
OTtest-G1-2F CGAAAAATGGTTAGTGGTGAC
OTtest-G1-2R GTGGATGTTACTGTAGCAATG
OTtest-SG1-1F AGAGGACCAAACACATTAACC
OTtest-SG1-1R TTTGGGGGAGAGCACTTTTG
OTtest-SG1-2F ATCACCTTGCTACTCTTTCTG
OTtest-SG1-2R GTTTAGTTGACCAAGCATCTC

Fig. 3

Result of gene editing vector restriction enzyme digestion of Gossypium barbadense L. A: GbU6-5P-GGB-sgRNA 1 editing vector digestion test results; M1 is 1 kb plus marker. B: GbU6-5P::GGB-sgRNA1-GbU6- 4P::GGB-sgRNA2 and GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2 edit vector restriction test results; M2 is 2 kb plus II marker."

Fig. 4

Protoplast preparation and transformation results of Gossypium barbadense L. A: microscopic examination of Gossypium barbadense L. protoplast before transformation; B: after protoplast microscopy results transformation of G. barbadense GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I vector. Bar = 40 μm."

Fig. 5

Transformation of different target sequence editing effect test results of Gossypium barbadense L. A: 1 is the result of direct PCR before transformation of GbU6-5P-GGB-sgRNA1-Cas9I vector, 2 is PCR amplification after transformation of GbU6-5P-GGB-sgRNA1-Cas9I vector As a result, 3 was obtained by direct PCR amplification before transformation of GbU6-5P-sgRNA1- Cas9I vector and 4 was PCR amplification after transformation with GbU6-5P-sgRNA1-Cas9I vector. B: 1 was transformed with GbU6- 5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I direct PCR amplification results, 2 for the transformation of GbU6-5P-sgRNA1-GbU6- 4P-sgRNA2-Cas9I direct PCR amplification results. C: G1 and E1 are the results of direct PCR before transformation of GbU6-5P:: GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I, G2 and E2 are the results of transformation of GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2-Cas9 digestion after PCR amplification results, G3 and E3 to transform GbU6-5P-sgRNA1-GbU6-4P-sgRNA2-Cas9I digestion before direct PCR amplification results, G4 and E4 to transform GbU6-5P-sgRNA1-PCR amplification results after GbU6-4P-sgRNA2-Cas9 digestion. M1 is 1 kb plus marker, M2 is 2 kb plus II marker."

Fig. 6

Sequencing results of base mutation of different types of target sequences of Gossypium barbadense L. A: Transformation of GbU6-5P-GGB-sgRNA 1-Cas9I vector target site editing effect sequencing detection results; B: Transformation GbU6- 5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I vector GGB target site editing effect sequencing detection results; C: GbU6-5P::GGB- sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I vector ERA1 target site editing sequencing results; D: transformation GbU6-5P::GGB-sgRNA1-GbU6- 4P::GGB-sgRNA2-Cas9I vector GGB-sgRNA2 target site editing effect sequencing test results; E: transformation GbU6-5P::GGB-sgRNA1-GbU6- 4P::GGB-sgRNA2-Cas9I vector GGB-sgRNA1 target site editing effect sequencing test results. The red box is the location of the PAM site."

Fig. 7

Sequencing peak map of base mutation of different types of target sequences of Gossypium barbadense L. A: baseline mutation map of genomes transformed with GbU6-5P-GGB-sgRNA1-Cas9I target sequence; B: transformation of GbU6- 5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I target sequence; C: baseline mutation sequencing of the genome of transformed GbU6- 5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I target sequence."

Table 4

Statistical results of editing efficiency of different target sequences"

转化载体名称
Conversion carrier name		 一个位点碱基突变个数
A base muta- tion number		 2个位点同时突变个数
Number of mutations for two sites at the same time		 突变率
Mutation rate
sgRNA1位点
sgRNA1 site		 sgRNA2位点
sgRNA2 site		 sgRNA1位点sgRNA1 site sgRNA2位点sgRNA2 site
GGB-sgRNA1 12/95 — — 0.126 —
Shift GGB-sgRNA1 10/95 — — 0.105 —
GGB-sgRNA1-ERA1-sgRNA2 — 15/70 15/70 0.214 0.214
Shift GGB-sgRNA1-ERA1-sgRNA2 — 11/70 10/70 0.157 0.143
GGB-sgRNA1-GGB-sgRNA2 — 9/57 7/57 0.157 0.123
Shift GGB-sgRNA1-GGB-sgRNA2 — 7/60 5/60 0.117 0.083

Fig. 8

Semi-quantitative detection of protoplast Cas9 and corresponding sgRNA in different types of target sequences transformed in Gossypium barbadense L. 1 and 2 is transformed GbU6-5P-GGB-sgRNA1-Cas9I edit vector and Control edit vector were detected GGB-sgRNA1, 3 and 4 is transformed GbU6-5P-GGB-sgRNA1-GbU6-4P::ERA1-sgRNA1- Cas9I edit vector were detected GGB-sgRNA1, ERA1-sgRNA2, 5 and 6 is transformed GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB- sgRNA2-Cas9I edit vector were detected GGB-sgRNA1, GGB- sgRNA2, 7 is transformed GbU6-5P-sgRNA1-4P-sgRNA1-Cas9I control edit vector were detected GGB-sgRNA1."

Fig. 9

Off target sequence amplification test of Gossypium barbadense L. Transform No shift sgRNA type target sequence: 1 and 2 is transformed GbU6-5P-GGB-sgRNA1-Cas9I edit vector and Control edit vector, 3 and 4 is transformed GbU6-5P-GGB-sgRNA1-GbU6- 4P::ERA1-sgRNA1-Cas9I edit vector and Control edit vector, 5 and 6 is transformed GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB-sgRNA2- Cas9I edit vector and Control edit vector. Transform Shift sgRNA type target sequence: 1 and 2 is transformed Shift GbU6-5P- GGB-sgRNA1-Cas9I edit vector and Control edit vector, 3 and 4 is transformed Shift GbU6-5P-GGB-sgRNA1-GbU6-4P::ERA1-sgRNA1- Cas9I edit vector and Control edit vector, 5 and 6 is transformed Shift GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I edit vector and Control edit vector."

Fig. 10

Off target sequence mutation detection of Gossypium barbadense L. A: OTtest-SG1-2 primer detection; 1 is transformed Shift GbU6- 5P-GGB-sgRNA1-Cas9I edit vector, 2 is transformed Shift GbU6- 5P-GGB-sgRNA1-GbU6-4P::ERA1-sgRNA1-Cas9I edit vector, 3 is transformed Shift GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB-sgRNA2- Cas9I edit vector. B:OTtest-G1-2 primer detection; 1 is transformed GbU6-5P-GGB-sgRNA1-Cas9I edit vector, 2 is transformed GbU6- 5P-GGB-sgRNA1-GbU6-4P::ERA1-sgRNA1-Cas9I edit vector, 3 is transformed GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I edit vector. C: OTtest-SG1-2 primer detection; 1 is transformed Shift GbU6-5P-GGB-sgRNA1-Cas9I edit vector, 2 is transformed Shift GbU6-5P-GGB-sgRNA1-GbU6-4P::ERA1-sgRNA1-Cas9I edit vector, 3 is transformed Shift GbU6-5P-GGB-sgRNA1-GbU6-4P::GGB-sgRNA2- Cas9I ditvector."

Fig. 11

Off target sequence sequencing peak map of Gossypium barbadense L. A, B, and C are OTtest-SG1-2, OTtest-G1-2, and OTtest-SG1-1 primers to transform the sequencing result of the target fragment amplified by the corresponding vector in the figure below, respectively. The red box identifies the base mutation site."

Table 5

Statistics of off-target efficiency of different target sequences"

转化载体名称
Conversion carrier name		 脱靶序列检测Detection of off-target sequences
OTtest-G1-1 OTtest-SG1-1 OTtest-SG1-2
GGB-sgRNA1 6/50 — —
Shift GGB-sgRNA1 — 0 18/50
GGB-sgRNA1-ERA1-sgRNA2 0 — —
Shift GGB-sgRNA1-ERA1-sgRNA2 — 0 6/50
GGB-sgRNA1-GGB-sgRNA2 0 — —
Shift GGB-sgRNA1-GGB-sgRNA2 — 0 6/50
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