Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (10): 233-242.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0475
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LI Xin-ge1,2(
), WANG Mei-xia2,3, WANG Chen-yang2, MA Gui-gen2,4, ZHOU Chang-yong3, WANG Ya-nan1(), ZHOU Huan-bin2,4,5()
Received:2024-05-21
Online:2024-10-26
Published:2024-11-20
Contact:
WANG Ya-nan, ZHOU Huan-bin
E-mail:xingell@hotmail.com;wyn3215347@163.com;hbzhou@ippcaas.cn
LI Xin-ge, WANG Mei-xia, WANG Chen-yang, MA Gui-gen, ZHOU Chang-yong, WANG Ya-nan, ZHOU Huan-bin. Development and Optimization of Genome Editing in Rice with CRISPR/LanCas9 System[J]. Biotechnology Bulletin, 2024, 40(10): 233-242.
| 引物名称Primer name | 序列Sequence（5'-3'） | 用途Application |
|---|---|---|
| OsMPK8-F1 | TGGCGGTGGAGGAGAGGTGA | Amplify the OsMPK8 target site in this research |
| OsMPK8-R1 | CAACCACCCATGACCATGAG | |
| OsWRKY45-F1 | TGCAGCCGCTCAAGGTGGAG | Amplify the OsWRKY45 target site in this research |
| OsWRKY45-R1 | TCAAACCCATAATGTCGTCC | |
| OsCPK6-F1 | AGAAACAGCATCTCTTGAGG | Amplify the OsCPK6 target site in this research |
| OsCPK6-R1 | GCTACTTGTTGCTGTGCTTG | |
| OsCPK4-F1 | TTCTCATCCCACACTGCGAC | Amplify two OsCPK4 target sites in this research |
| OsCPK4-R1 | CCGAAATGGAACTCCCGAAT | |
| OsCPK7-F1 | CCTGGGAAGGATACAATTGT | Amplify the OsCPK7 target site in this research |
| OsCPK7-R1 | TAGCTATCCCCCTGAACGTA | |
| OsGSK3-F1 | TGTTCTGCCTTGCCTCACTGG | Amplify the OsGSK3 target site in this research |
| OsGSK3-R1 | ACACTGCAGAAGTTTACCTG | |
| OsGSK4-F1 | GGACAAAGAATGTCGCATGA | Amplify the OsGSK4 target site in this research |
| OsGSK4-R1 | ACATGCACAAGACTAATACG | |
| gOsMPK8-F1 | tgttgCGGAGGAAGGTGAGAGGAGG | Target the OsMPK8（20-nt）target site |
| gOsMPK8-R1 | aaacCCTCCTCTCACCTTCCTCCGc | |
| gOsMPK8-F2 | tgttgCAGACGGAGCAGCAGCAGCAGCAG | Target the OsMPK8（24-nt）target site |
| gOsMPK8-R2 | aaacCTGCTGCTGCTGCTGCTCCGTCTGc | |
| gOsWRKY45-F1 | tgttgTGGAGCTACGACGCCGTCGC | Target the OsWRKY45（20-nt）target site |
| gOsWRKY45-R1 | aaacGCGACGGCGTCGTAGCTCCAc | |
| gOsWRKY45-F2 | tgttgGCACTGGAGCTACGACGCCGTCGC | Target the OsWRKY45（24-nt）target site |
| gOsWRKY45-R2 | aaacGCGACGGCGTCGTAGCTCCAGTGCc | |
| gOsCPK6-F1 | gtgtgATGGGCAACTACTACTCGTG | Target the OsCPK6 target site |
| gOsCPK6-R1 | aaacCACGAGTAGTAGTTGCCCATc | |
| gOsCPK4-F1 | tgttGCGGGAGCGGGAAGCGGCAG | Target the OsCPK4 T1 target site |
| gOsCPK4-R1 | aaacCTGCCGCTTCCCGCTCCCGC | |
| gOsCPK4-F2 | gtgtgTCGCCGTCAAGCGCATCGAC | Target the OsCPK4 T2 target site |
| gOsCPK4-R2 | aaacGTCGATGCGCTTGACGGCGAc | |
| gOsCPK7-F1 | tgttgTGCCAGAACGGGACTCTTGG | Target the OsCPK7 target site |
| gOsCPK7-R1 | aaacCCAAGAGTCCCGTTCTGGCAc | |
| gOsGSK3-F1 | gtgtGGAAGAATGGAGAACCTAAA | Target the OsGSK3 target site |
| gOsGSK3-R1 | aaacTTTAGGTTCTCCATTCTTCC | |
| gOsGSK4-F1 | tgttgAACCTGGAATACAACACCAA | Target the OsGSK4 target site |
| gOsGSK4-R1 | aaacTTGGTGTTGTATTCCAGGTTc | |
| g4-F1 | TAAGCTTGATATCGAATTCG | Amplify the PENTR4:gRNA4 vector to construct the PENTR4:gRNA34 plasmid |
| g4-R1 | TAGCCAACACAAGCGGCAGC | |
| g34-F1 | CGCTGCCGCTTGTGTTGGCTAGGATCCATCGCAGTCAGCG | Amplify the sgRNA fragments to construct the PENTR4:gRNA34 plasmid |
| g34-R1 | CGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG |
Table 1 Primer sequences used in this study
| 引物名称Primer name | 序列Sequence（5'-3'） | 用途Application |
|---|---|---|
| OsMPK8-F1 | TGGCGGTGGAGGAGAGGTGA | Amplify the OsMPK8 target site in this research |
| OsMPK8-R1 | CAACCACCCATGACCATGAG | |
| OsWRKY45-F1 | TGCAGCCGCTCAAGGTGGAG | Amplify the OsWRKY45 target site in this research |
| OsWRKY45-R1 | TCAAACCCATAATGTCGTCC | |
| OsCPK6-F1 | AGAAACAGCATCTCTTGAGG | Amplify the OsCPK6 target site in this research |
| OsCPK6-R1 | GCTACTTGTTGCTGTGCTTG | |
| OsCPK4-F1 | TTCTCATCCCACACTGCGAC | Amplify two OsCPK4 target sites in this research |
| OsCPK4-R1 | CCGAAATGGAACTCCCGAAT | |
| OsCPK7-F1 | CCTGGGAAGGATACAATTGT | Amplify the OsCPK7 target site in this research |
| OsCPK7-R1 | TAGCTATCCCCCTGAACGTA | |
| OsGSK3-F1 | TGTTCTGCCTTGCCTCACTGG | Amplify the OsGSK3 target site in this research |
| OsGSK3-R1 | ACACTGCAGAAGTTTACCTG | |
| OsGSK4-F1 | GGACAAAGAATGTCGCATGA | Amplify the OsGSK4 target site in this research |
| OsGSK4-R1 | ACATGCACAAGACTAATACG | |
| gOsMPK8-F1 | tgttgCGGAGGAAGGTGAGAGGAGG | Target the OsMPK8（20-nt）target site |
| gOsMPK8-R1 | aaacCCTCCTCTCACCTTCCTCCGc | |
| gOsMPK8-F2 | tgttgCAGACGGAGCAGCAGCAGCAGCAG | Target the OsMPK8（24-nt）target site |
| gOsMPK8-R2 | aaacCTGCTGCTGCTGCTGCTCCGTCTGc | |
| gOsWRKY45-F1 | tgttgTGGAGCTACGACGCCGTCGC | Target the OsWRKY45（20-nt）target site |
| gOsWRKY45-R1 | aaacGCGACGGCGTCGTAGCTCCAc | |
| gOsWRKY45-F2 | tgttgGCACTGGAGCTACGACGCCGTCGC | Target the OsWRKY45（24-nt）target site |
| gOsWRKY45-R2 | aaacGCGACGGCGTCGTAGCTCCAGTGCc | |
| gOsCPK6-F1 | gtgtgATGGGCAACTACTACTCGTG | Target the OsCPK6 target site |
| gOsCPK6-R1 | aaacCACGAGTAGTAGTTGCCCATc | |
| gOsCPK4-F1 | tgttGCGGGAGCGGGAAGCGGCAG | Target the OsCPK4 T1 target site |
| gOsCPK4-R1 | aaacCTGCCGCTTCCCGCTCCCGC | |
| gOsCPK4-F2 | gtgtgTCGCCGTCAAGCGCATCGAC | Target the OsCPK4 T2 target site |
| gOsCPK4-R2 | aaacGTCGATGCGCTTGACGGCGAc | |
| gOsCPK7-F1 | tgttgTGCCAGAACGGGACTCTTGG | Target the OsCPK7 target site |
| gOsCPK7-R1 | aaacCCAAGAGTCCCGTTCTGGCAc | |
| gOsGSK3-F1 | gtgtGGAAGAATGGAGAACCTAAA | Target the OsGSK3 target site |
| gOsGSK3-R1 | aaacTTTAGGTTCTCCATTCTTCC | |
| gOsGSK4-F1 | tgttgAACCTGGAATACAACACCAA | Target the OsGSK4 target site |
| gOsGSK4-R1 | aaacTTGGTGTTGTATTCCAGGTTc | |
| g4-F1 | TAAGCTTGATATCGAATTCG | Amplify the PENTR4:gRNA4 vector to construct the PENTR4:gRNA34 plasmid |
| g4-R1 | TAGCCAACACAAGCGGCAGC | |
| g34-F1 | CGCTGCCGCTTGTGTTGGCTAGGATCCATCGCAGTCAGCG | Amplify the sgRNA fragments to construct the PENTR4:gRNA34 plasmid |
| g34-R1 | CGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAG |
Fig. 1 Schematics of vector A: Schematics of LanCas9 expression components. B: Schematics of SLanCas9 expression components. C: Schematics of pENTR4-gRNA34 vector
| 基因Gene | 基因登录号Gene identifier | 间隔区Spacer | PAM序列PAM sequence |
|---|---|---|---|
| OsWRKY45 | LOC_Os05g25770 | TGGAGCTACGACGCCGTCGC | CGG |
| TGCACTGGAGCTACGACGCCGTCGC | CGG | ||
| OsCPK4 T1 | LOC_Os02g03410 | GCGGGAGCGGGAAGCGGCAG | CAG |
| OsCPK4 T2 | LOC_Os02g03410 | TCGCCGTCAAGCGCATCGAC | AAG |
| OsCPK6 | LOC_Os02g58520 | ATGGGCAACTACTACTCGTG | CGG |
| OsCPK7 | LOC_Os03g03660 | TGCCAGAACGGGACTCTTGG | GAG |
| OsMPK8 | LOC_Os03g59390 | CGGAGGAAGGTGAGAGGAGG | TGG |
| CAGACGGAGCAGCAGCAGCAGCAG | CGG | ||
| OsGSK3 | LOC_Os02g14130 | GGAAGAATGGAGAACCTAAA | AGG |
| OsGSK4 | LOC_Os06g35530 | AACCTGGAATACAACACCAA | AGG |
Table 2 Target genes in this study
| 基因Gene | 基因登录号Gene identifier | 间隔区Spacer | PAM序列PAM sequence |
|---|---|---|---|
| OsWRKY45 | LOC_Os05g25770 | TGGAGCTACGACGCCGTCGC | CGG |
| TGCACTGGAGCTACGACGCCGTCGC | CGG | ||
| OsCPK4 T1 | LOC_Os02g03410 | GCGGGAGCGGGAAGCGGCAG | CAG |
| OsCPK4 T2 | LOC_Os02g03410 | TCGCCGTCAAGCGCATCGAC | AAG |
| OsCPK6 | LOC_Os02g58520 | ATGGGCAACTACTACTCGTG | CGG |
| OsCPK7 | LOC_Os03g03660 | TGCCAGAACGGGACTCTTGG | GAG |
| OsMPK8 | LOC_Os03g59390 | CGGAGGAAGGTGAGAGGAGG | TGG |
| CAGACGGAGCAGCAGCAGCAGCAG | CGG | ||
| OsGSK3 | LOC_Os02g14130 | GGAAGAATGGAGAACCTAAA | AGG |
| OsGSK4 | LOC_Os06g35530 | AACCTGGAATACAACACCAA | AGG |
| PAM序列 PAM Seq | 基因 Gene | 打靶位点 Target site | Spacer长度 Spacer lengths/bp | 突变效率 Mutation efficiency | 单等位突变 Mono-allelic mutation | 双等位突变 Bi-allelic mutation |
|---|---|---|---|---|---|---|
| NGG | OsMPK8 | CGGAGGAAGGTGAGAGGAGGTGG | 20 | 5/48（10.42%） | 5 | 0 |
| CAGACGGAGCAGCAGCAGCAGCAGCGG | 24 | 1/48（2.08%） | 1 | 0 | ||
| OsWRKY45 | TGGAGCTACGACGCCGTCGCCGG | 20 | 12/48（25.00%） | 11 | 1 | |
| TGCACTGGAGCTACGACGCCGTCGCCGG | 24 | 2/48（4.17%） | 2 | 0 |
Table 3 Efficiency of gene editing by LanCas9 in rice
| PAM序列 PAM Seq | 基因 Gene | 打靶位点 Target site | Spacer长度 Spacer lengths/bp | 突变效率 Mutation efficiency | 单等位突变 Mono-allelic mutation | 双等位突变 Bi-allelic mutation |
|---|---|---|---|---|---|---|
| NGG | OsMPK8 | CGGAGGAAGGTGAGAGGAGGTGG | 20 | 5/48（10.42%） | 5 | 0 |
| CAGACGGAGCAGCAGCAGCAGCAGCGG | 24 | 1/48（2.08%） | 1 | 0 | ||
| OsWRKY45 | TGGAGCTACGACGCCGTCGCCGG | 20 | 12/48（25.00%） | 11 | 1 | |
| TGCACTGGAGCTACGACGCCGTCGCCGG | 24 | 2/48（4.17%） | 2 | 0 |
Fig. 2 Editing patterns of OsMPK8 and OsWRKY45 induced by LanCas9 A: Editing pattern of OsMPK8 induced by LanCas9 with 20-nt spacer. B: Editing pattern of OsWRKY45 induced by LanCas9 with 20-nt spacer. C: Editing pattern of OsMPK8 induced by LanCas9 with 24-nt spacer. D: Editing pattern of OsWRKY45 induced by LanCas9 with 24-nt spacer. The PAM sequences, target sequences, and detected nucleotide changes are highlighted in green, bold, and red, respectively; WT: wild type
| PAM序列PAM Seq | 基因Gene | 突变效率Mutation efficiency | 单等位基因突变Mono-allelic mutation | 双等位基因突变Bi-allelic mutation |
|---|---|---|---|---|
| NGG | OsWRKY45 | 25/31（80.65%） | 2 | 23 |
| OsCPK6 | 48/48（100.00%） | 5 | 43 | |
| NAG | OsCPK4 T1 | 19/48（39.58%） | 18 | 1 |
| OsCPK7 | 0/48（0.00%） | 0 | 0 |
Table 4 Efficiency of gene editing by SLanCas9 in rice
| PAM序列PAM Seq | 基因Gene | 突变效率Mutation efficiency | 单等位基因突变Mono-allelic mutation | 双等位基因突变Bi-allelic mutation |
|---|---|---|---|---|
| NGG | OsWRKY45 | 25/31（80.65%） | 2 | 23 |
| OsCPK6 | 48/48（100.00%） | 5 | 43 | |
| NAG | OsCPK4 T1 | 19/48（39.58%） | 18 | 1 |
| OsCPK7 | 0/48（0.00%） | 0 | 0 |
Fig. 3 Representative mutations in OsCPK6 and OsCPK4 T1 genes in rice by SLanCas9 The PAM sequences, target sequences, and detected nucleotide changes are highlighted in green, bold, and red, respectively; WT: wild type
Fig. 4 Editing type and proportion of SLanCas9 in rice's genes Bi: Bi-allelic mutation; Mono: mono-allelic mutation; Ho: homozygotes; He: heterozygotes; Del: deletion; WT: wild type
| PAM序列PAM Seq | 基因Gene | 突变效率Mutation efficiency | 单等位基因突变Mono-allelic mutation | 双等位基因突变Bi-allelic mutation |
|---|---|---|---|---|
| NGG | OsGSK3 | 21/27（77.78%） | 20 | 1 |
| OsGSK4 | 26/27（96.30%） | 10 | 16 | |
| NAG | OsCPK4 T1 | 14/48（29.17%） | 14 | 0 |
| OsCPK4 T2 | 0/48（0.00%） | 0 | 0 |
Table 5 Efficiency of SLanCas9 in multiple gene editing in rice
| PAM序列PAM Seq | 基因Gene | 突变效率Mutation efficiency | 单等位基因突变Mono-allelic mutation | 双等位基因突变Bi-allelic mutation |
|---|---|---|---|---|
| NGG | OsGSK3 | 21/27（77.78%） | 20 | 1 |
| OsGSK4 | 26/27（96.30%） | 10 | 16 | |
| NAG | OsCPK4 T1 | 14/48（29.17%） | 14 | 0 |
| OsCPK4 T2 | 0/48（0.00%） | 0 | 0 |
Fig. 5 Mutational events of SLanCas9 in OsGSK3/OsGSK4 multiplex gene editing Del: Delate; Insert: insert; WT: wild type
Fig. 6 The editing type and proportion of SLanCas9 in OsGSK3/OsGSK4 multiple gene editing Bi: Bi-allelic mutation; Mono: mono-allelic mutation; Ho: homozygotes; He: heterozygotes; Del: deletion; WT: wild type
Fig. 7 Representative mutation in OsCPK4 T1 multiplex gene editing of SLanCas9 The PAM sequences, target sequences, and detected nucleotide changes are highlighted in green, bold, and red, respectively; WT: wild type
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