PB-LMluIT1OsU6aOsU6bT2OsU6cT3OsU3T4MluIGA-RPB-RGA-LLacZ
Figure 6. Generation of a 4-target construct by Gibson Assembly cloning
(1) 根据在一个载体构建靶点数的需要，合成以下引物对。 以下相同颜色表示连接配对序列，3’端黑色字母表示与U3/6启动子上游配对序列(U-GA#)或与sgRNA下游配对(Pgs-GA#)序列，长框为各种通用引物配对位点（标注在右侧），位于各表达盒的连接处，用于测序检验阳性克隆时，测序公司可以提供这些通用引物。
Table S4. Primers used for Gibson Assembly of sgRNA expression cassettes.
Position 1st PCR 2nd PCR Site 2 Site 2 Site 3 Site 3 Site 4 Site 4 Site 5 Site 5 Site 6 7 Site 7 Site 8 Site 8
Primer U-F gR-R
Pgs-GA2 U-GA2 Pgs-GA3 U-GA3 Pgs-GA4 U-GA4 Pgs-GA5 U-GA5 Pgs-GA6 Sequence (5’--3’)
CTCCGTTTTACCTGTGGAATCG CGGAGGAAAATTCCATCCAC
ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG Ptac promoter F primer site GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG Sp6 promoter primer site CGCTATAGTGTCACCTAAATCGTATGCGTGGTGGAATCGGCAGCAAAGG GTCGCTAGTTATTGCTCAGCGGCCAAGCTCATCCACTCCAAGCTCTTG T7 terminator F primer site GAGCTTGGCCGCTGAGCAATAACTAGCGACTGGAATCGGCAGCAAAGG
CATCGTCGCCGTCCAGCTCGACCATTGAACATCCACTCCAAGCTCTTG EGFP-N primer site GTTCAATGGTCGAGCTGGACGGCGACGATGTGGAATCGGCAGCAAAGG GCTCCGAATACGACTCACTATAGGGTGACCATCCACTCCAAGCTCTTG T7 universal primer site GGTCACCCTATAGTGAGTCGTATTCGGAGCTGGAATCGGCAGCAAAGG CTGAGGTTAACCCTCACTAAAGGGAAGCTCCATCCACTCCAAGCTCTTG T3 Promoter primer site GGAGCTTCCCTTTAGTGAGGGTTAACCTCAGTGGAATCGGCAGCAAAGG
CGTGGTATGCTAGTTATTGCTCAGCCTCGACATCCACTCCAAGCTCTTG T7 terminator R primer site GTCGAGGCTGAGCAATAACTAGCATACCACGTGGAATCGGCAGCAAAGG TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGCTCTTG Site B-L U-GAL Site 6 Site U-GA6
Pgs-GA7 U-GA7 Pgs-GA8 U-GA8
Site B-R Pgs-GAR Note:
(1) One or multiple sgRNA expression cassettes are amplified with the primer pairs in this way: 1 cassette: Pps-GAL/Pgs-GAR;
2 cassettes: Pps -GAL/Pgs-GA2, Pps-GA2/Pgs-GAR;
3 cassettes: Pps-GAL/Pgs-GA2, Pps-GA2/Pgs-GA3, Pps-GA3/Pgs-GAR;
4 cassettes: Pps-GAL/Pgs-GA2, Pps-GA2/Pgs-GA3, Pps-GA3/Pgs-GA4, Pps-GG4/Pgs-GAR; and so on.
(2) The same flanking primers PB-L and PB-R shown in Table S1 can be used to amplify, if necessary, the
ligated sgRNA expression cassettes.
注：为方便操作，参考11页（6）模式预先混合好引物组。
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（2）自制Isothermal in vitro recombination reaction mixture：
5X isothermal（ISO）reaction buffer：25% PEG-8000，500 mmol/L Tris-HCl pH 7.5，50 mmol/L MgCl2，50 mmol/L DTT，4种dNTPs 各1 mmol/L，5 mmol/L NAD，?20 °C保存。
2 X master mixture：200 μL 5 × ISO buffer, T5 Exonuclease (Epicentre) 2.0 units（严格控制T5 exouclease酶量，不能过少和过多！过多T5 exouclease会造成DNA片段被快速降解），Phusion High-Fidelity DNA polymerase (NEB) 25 units, Taq ligase (NEB) 2500 units, deionized water to 0.5 ml。多管分装?20 °C保存.
（3）参考第8页指引设计U3/6启动子排列。但如果是4个靶点，建议把较长的U6c用于T3, U6b用于T4,这样可以用SP2测通T4和T3。
（4）在50 μl反应用20 U Bsa I酶切~2 μg pYLCRISPR/Cas9 约30 min。取2μl（~80 ng）电泳检查，确认被切出的ccdB条带（732 bp）。65℃5min失活，每管3 μl（~100 ng）约分装冷冻保存（一般不需要回收纯化）。
（5） 按以上策略I步骤(2)至步骤(5)的第一轮PCR同样操作；第二轮PCR使用以上Table S4引物对(各引物0.15μM)扩增各靶点（T1, T2, T3,...）的gRNA表达盒片段。取2 μl电泳检查。
（6）根据各样品产物估算的量，把所有产物大致等量混合，酚抽提乙醇沉淀或用PCR产物纯化kit纯化；或在PCR产物中加入单链核酸酶Exonuclease I (ExoI, NEB) 0.5 μl (10 units)在37°C处理10-15 min消除剩余引物（这是为了避免引物与目的片段竞争结合），80°C 20min失活ExoI（以避免消化Gibson反应中产生的单链末端），不需纯化直接取适量进入步骤7。
（7）在分装有切好的pYLCRISPR/Cas9（~100 ng）的管中加入适量的gRNA表达盒片段(每表达盒~15ng)，加入去离子水至最终量7~8 μl。加入等量7~8 μl 2X master mixture，50 °C反应25 ~30 min（时间过长可能会造成DNA被T5 exouclease降解！）。可以取10 μl连接产物电泳检查，正常反应可见连接的较大片段（见18页图）。
（8）参考策略I步骤(12)操作，但在透析膜对0.2 x TE透析较短时间的5-10 min（这是因为ISO buffer含有PEG-8000，过长时间透析会吸收过多的0.2 x TE降低连接产物浓度。但不透析直接电激转化容易打炸或电激电流过大而降低转化效率）。
（9）阳性克隆PCR检测按按策略I步骤(13)进行；测序策略也可以用Table S4所示的通用引物（测序公司可提供，将这些测序引物序列交测序公司确认）,从各表达盒连接点往sgRNA反向进行测序。每个引物可以测通2个靶点。
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附录2：载体构建相关电泳图
Construction of a CRISPR/Cas9 vector with four targeting sites0.5 kb23 kb9 kb1 kb0.5 kb15 kb3,Colony PCR screening for positive clonesCas9vector backboneLinked 4 gRNAexpression cassettes1,Recovered Cas9vector backbone digested with BsaI2, PCR amplification of four gRNAexpression cassettes 3 kbChecked clones4,Restriction analysis of positive clones with AscICas9 vector backboneLinked 4 gRNAexpression cassettes5,Stability verification of the Cas9construct in Agrobacteriumtumefaciens(the plasmid from Agrobacteriumwas transferred back to E. coli, then clone plasmids were isolated and digested with AscI)
Construction of a CRISPR/Cas9 vector with eight targeting sites2 kb0.5 kb1, PCR amplification of eight gRNAexpression cassettes (first round of amplification)23 kb4.3 kbCas9vector backboneLinked 8 gRNAexpression cassettes2,Restriction analysis of positive clones with AscI1 kb0.5 kb3, Testing one positive clone with PCR, using pairs of the target adaptor primers(F, R are forward and reverse primers, respectively).Cas9vector backboneLinked 8 gRNAexpression cassettes4,Stability verification of the Cas9construct in Agrobacteriumtumefaciens, clones were digested with AscI
（以上为Golden Gate方法构建）
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A 6-target CRISPR/Cas9 construct prepared by Gibson assembly
附录3：载体序列
GenBank data library under accession numbers: KR029097, KR029098, KR029099, KR0290100, KR0290101, KR0290102, KR0290103, KR0290104, KR0290105, KR0290106, KR0290107, KR029108 for the CRISPR/sgRNA intermediate plasmids, and KR0290109, KR0290110, KR0290111, KR0290112, KR0290113 for the CRISPR/Cas9 binary vectors.
LacZ-OsU6a-sgRNA structure in the plasmidBsaIBamHIacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGACGCGTTGAMluIE.coliPromoter LacZ(alpha)CATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaa tcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagtt gcgcagcctgaatggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18 backbone）BsaIHindIII
PCR product of LacZ-U6a-sgRNA (831 bp, 801 bp after Bsa I digestion)
TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaaTTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT
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OsU6a-sgRNA structure in the plasmidBsaIBamHIacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone）Hind IIIBsaI
PCR product of OsU6a-sgRNA (629 bp, 599 bp after Bsa I digestion)
TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG(19-20 bp target)GTTTTAG
AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT GGAGTGGATGGNNNNNNNCGAGACCCACGCT
OsU6b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCCACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTGAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)
PCR product of OsU6b-sgRNA (515 bp, 485 bp after Bsa I digestion)
TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACG CT
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