植物多靶点CRISPR-Cas9载体使用方法(2015-5-1) - 图文

CRISPR/Cas9-based genome editing technology

A robust CRISPR/Cas9 vector system for multiplex targeting of

genomic sites in monocot and dicot plants

亚热带农业生物资源保护与利用国家重点实验室

华南农业大学生命科学学院

刘耀光课题组

(ygliu@scau.edu.cn)

1. pYLCRISPR/Cas9多靶点载体介绍

CRISPR/Cas9是新近发展的基因组编辑技术（图1）。CRISPR/Cas9切割靶序列仅需要single-guide RNA (sgRNA)以及由sgRNA引导的Cas9蛋白，比锌指核酸酶（ZFNs），TALENs更加简便，高效，因而成为基因组编辑工具的首选。

我们利用CRISPR/Cas9技术可方便地进行多重靶向的特征，构建了一套用于单子叶和双子叶植物的多靶点CRISPR/Cas9基因打靶载体系统。

本套载体将Cas9蛋白表达盒整合到双元载体上，用于装载多个sgRNA表达盒的多克隆位点Bsa I位于靠近双元载体RB位置。sgRNA表达盒元件设在中间质粒载体上，利用酶切连接和PCR方法拼装好，再利用Golden Gate或Gibson Assembly克隆方法组装到双元载体上。本载体系统已经发表（Ma et al., 2015, Molecular Plant, DOI:10.1016/j.molp.2015.04.007）。

Cas9 nucleaseRuvC-like domainHNH domainTarget Site PAMNGGNNNNNNNNNNNN-3’genome NCCNNNNNNNNNNNN-5’sequenceAA5’ NNNNNNNNNNNN3’ NNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNCleavage siteNNNNNNNNNNNNNNNNNNNN5’-G/ANNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAACUAUUGCCUGAUCGGAAUAAAAUUCGAUAGAAFigure 1. A working model of the CRISPR/Cas9 system. The Cas9-sgRNA complex locates to the target site to cleave the DNA to produce double strand break (DSB).

1

CUUGAAAAAGUGGCACCGAUUUUU-3’GCGUGGCU

2．CRISPR/Cas9载体与sgRNA载体图谱

2.1CRISPR/Cas9双元载体

本套载体系统的双元载体骨架为pCAMBIA-1300 (ACCESSION: AF234296)，Cas9p为本实验室设计合成的植物优化密码子基因，它模拟了禾本科植物基因具有5’端GC含量较高的特征 (Figure 2)。

这些质粒在E. coli TOP10F’(LacIq) 菌株繁殖，该菌株LacIq基因型产生的阻碍蛋白可抑制ccdB大肠杆菌致死基因的表达。

我们对pYLCRISPR/Cas9载体采用了新的命名，与旧的命名对应关系见表1。

pYLCRISPR/Cas9Pubi-H, -B载体中的Cas9p用PUbi驱动，优选用于单子叶植物的基因打靶；对于双子叶植物，我们前期使用pYLCRISPR/Cas9P35S-H在拟南芥获得了uniform简单突变（双等位突变，杂合突变，纯合突变）以及嵌合突变（Ma et al., 2015）。但是，我们最近发现用pYLCRISPR/Cas9Pubi-H在拟南芥的打靶效果效率比使用pYLCRISPR/Cas9P35S-H的效果效率更高，能得到更多的简单突变。因此也推荐在双子叶植物优先使用PUbi驱动Cas9p的载体pYLCRISPR/Cas9P35S-H或pYLCRISPR/Cas9P35S-B。

Figure 2. A CRISPR/Cas9 system for monocot and dicot plants.

pYLCRISPR/Cas9Pubi-H, -B (above); pYLCRISPR/Cas9P35S-H, -N,-B (below); HPT (-H), Bar (-B), and NPT II (-N) encode hygromycin B phosphotransferase, PPT acetyltransferase and neomycin phosphotransferase II, respectively. NLS, nuclear localization sequence. The key sequences including restriction sites for cloning and analysis of sgRNA expression cassettes are given. Two Bsa I-cutting sties [Bsa I (B-L) and Bsa I (B-R)] for cloning the sgRNA expression cassettes are

separated by a shorten form of the ccdB negative selection gene.

2

表一 pYLCRISPR/Cas9载体新旧命名对应关系 新命名

pYLCRISPR/Cas9Pubi-H

原命名

pYLCRISPR/Cas9-MH, pYLCRISPR/Cas9-MTmono

pYLCRISPR/Cas9Pubi-B pYLCRISPR/Cas9P35S-H pYLCRISPR/Cas9P35S-B pYLCRISPR/Cas9P35S-N

pYLCRISPR/Cas9-MB pYLCRISPR/Cas9-DH pYLCRISPR/Cas9-DB pYLCRISPR/Cas9-DN

单子叶/双子叶 双子叶 双子叶 双子叶

草甘磷 潮霉素 草甘磷 卡那霉素

适用植物种 单子叶/双子叶

植物抗性 潮霉素

2.2 CRISPR/sgRNA vectors

sgRNA序列全长97 nt，分为两部分，5’决定靶序列的20nt (seed sequence)和3’区域为保守的结构序列。因此构建针对特定靶位点的sgRNA，只需要克隆决定靶序列的5’端20 nt。 sgRNA用small nuclear (sn) RNA U6/U3启动子驱动，以保证转录出的sgRNA停留在细胞核中与Cas9结合。

本方案用PCR扩增的方法构建包含靶序列的sgRNA表达盒，并在扩增引物5’端引入顺次排列的位置特异Bsa I 酶切位点，用于Golden Gate克隆。或在扩增引物5’端加入互补序列用于Gibson Assembly 连接克隆。

为了提高构建好的多靶点载体的稳定性，避免在农杆菌或者植物基因组中sgRNA表达盒之间发生同源重组，从水稻克隆了4个不同snRNA启动子(OsU3,OsU6a，OsU6b，OsU6c)，用于单子叶植物；从拟南芥中克隆了4个不同snRNA启动子（AtU3b，AtU3d，AtU6-1，AtU6-29），用于双子叶植物。

Figure 3．(A) Overall structure of eight basic sgRNA intermediate vectors. (B) The two Bsa I cutting

sequences are given in twelve sgRNA vectors (in a linear form), including other four vectors

3

(pYLsgRNA-OsU3/LacZ, pYLsgRNA-OsU6a/LacZ, pYLsgRNA-AtU3b/LacZ, and pYLsgRNA-AtU3d/LacZ) that have an additional LacZ gene (198 bp) as a cloning selection marker. The U3 and U6 promoters from rice (Os) and Arabidopsis (At) and the sgRNA sequence are separated by the vector backbone, to avoid amplification of the uncut plasmids by PCR with short extension time during the construction of sgRNA expression cassettes. Cutting (small arrows) with Bsa I produces different non-palindromic sticky ends to the promoters and an end to the sgRNA sequence. (C) A representative regular target and an irregular target, their target adaptors for the OsU6a promoter, and the transcribed 5’ sequences are shown. A ligated target-sgRNA expression cassette is amplified by nested PCR using primers U-F/gR-R and Pps/Pgs. Pps and Pgs are position-specific primers with Bsa I sites for the Golden Gate ligation (Table S1), or with overlapping ends for Gibson Assembly (Table S3).

3.靶位点选择和接头设计

3.1 靶位点选择

建议对1个目的基因设计2个靶点(尤其只有一个靶基因)，以降低某靶点打靶不成功的风险。可在ORF 5’区和功能结构域各设计1个靶点，使之任何1个靶点的突变都可以产生功能缺失，或2个靶点之间的序列被敲除。设计靶点的原则：

(1) 目的基因(转录方向)的正链和负链靶点的打靶效率大致相同，都可以设计靶点； (2) Regular targets & irregular targets (见以下说明)有同等打靶效率；

(3) 靶点的GC%尽量不要低于40%，靶点序列GC%偏高(50-70%)有较高的打靶效率。靶点内(按5-N20NGG-3

方向)不要有连续4个以上的T，以防RNA Pol III将其作为转录终止信号；

(4) 虽然非特异打靶（脱靶）对植物基因打靶不是很重要的问题，但应进行靶点特异性分析：用靶点+NGG

（前后各加几十碱基）与基因组做blast (选Somewhat similar sequences），避免使用与同源序列差异少于5个碱基的靶点(在切点附近和PAM可有2个碱基差异就具有特异性)。可以使用在线软件CRISPR-P

(http://cbi.hzau.edu.cn/crispr/)做这个分析，但可使用的靶点序列不一定限于Regular targets (AN19NGG或GN19NGG)，见以下关于Regular target & irregular target说明。

(5) 打算用一个靶序列敲除2个或以上的同源基因时，选择同源基因中碱基完全相同的区域为靶位点。 (6) 把靶点序列连接到sgRNA序列的5’端[(20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAA

GGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT],利用在线软件RNA Folding Form (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3)做二级结构分析。靶序列与sgRNA序列产生连续配对7 bp（注意：RNA可以产生U-G配对）以上会抑制其与染色体DNA靶序列结合靶点，因此要避免使用连续配对7 bp以上的靶序列。

3.2靶点接头设计

U6/U3基因由III型RNA聚合酶转录，转录起始点是确定的碱基位置。例如，U6为G碱基，U3为A碱基。因此，由U6/U3启动子驱动的sgRNA首碱基一定是G或者A。但是设计的靶序列的首碱基不一定刚好是G或者A，因此设计接头引物时需分两种情况考虑： （1）如果目标区NGG(PAM)上游第20碱基是A（用U3启动子）或G(用U6启动子），作为常规靶点(regular target)，将A/G下游19碱基可按下图所示合成接头。

4

19ntPAM5-NNNNNNNNN(A/G)NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNN-3target19nt合成接头的形式：5-ggcANNNNNNNNNNNNNNNNNNN-3’(forward adaptor primer, T#-F)（对OsU3启动子）3-NNNNNNNNNNNNNNNNNNNCAAA-5’(reverse adapter primer, T#-R)

常规靶点（regular target）接头（target adaptor）的形式

注：接头正向引物T#-F和反向引物T#-R命名是根据靶点在sgRNA表达盒的连接方向决定，不是基因方向决定，否则这些引物用于检测阳性克隆时容易搞错方向; 另外注意设计引物（尤其反向引物）时的5’-3’方向。

（2）如果NGG上游第20碱基不是A或G，作为非常规靶点(irregular target)，将20碱基为靶序列合成接头的形式

PAM5-NNNNNNNNN(T/C)NNNNNNNNNNNNNNNNNNNNGGNNNNNNNNN-320nt合成接头的形式：5-gtcA(T/C)NNNNNNNNNNNNNNNNNNN-3（对AtU3b启动子）3-(A/G)NNNNNNNNNNNNNNNNNNNCAAA-5非常规靶点（irregular target）接头的形式

也就是说，所用启动子的转录起始点与NGG上游第20碱基相同的regular靶点就是合成19碱基+4碱基粘性末端(转录产生的靶点配对序列为A/G+19 =20)；不相同的irregular靶点就是合成20碱基+4碱基粘性末端。

对使用拟南芥的AtU3/AtU6启动子的靶点接头也是同样道理，但对接各启动子的粘性末端不同（见Figure 3B）。

20nt

3.3 Overlapping PCR法构建sgRNA表达盒的引物设计 见4.1.2（与3.2相同目的，自由选择用任一个方案）

4. pYLCRISPR/Cas9打靶载体构建原理

4.1靶点引物接头与sgRNA表达盒的连接和扩增

4.1.1 sgRNA构建方法1 (接头连接扩增法，与3.2对应)：

连接接头后（连接操作见步骤5-5），有3种方法扩增sgRNA表达盒片段（Figure 4）：

（1）直接用Golden Gate位置特异引物(Table S1)做一轮PCR扩增。此方案效果不那么稳定，且不能避免扩增下图的产物IV和产物V。

（2）做2轮巢式PCR，第一轮PCR用通用的U-F/gR-R做1个反应（引物序列见附录），第二轮PCR用位置特异引物扩增。此方案扩增效果好且稳定，但同样不能避免扩增产物IV和产物V（注1）。

5

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。

16

（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。

17

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）

18

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

19

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

20

OsU6c-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGActcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaaaccaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatacttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacattatccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaataatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgacaccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatggaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtggtggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaacaacagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcagAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of OsU6c-gRNA (924 bp, 894 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGActcattagcggtatgcatgttggtagaagtcggagatgtaaataattttcattatataaaaaaggtacttcgagaaaaataaatgcatacgaattaattctttttatgttttttaaaccaagtatatagaatttattgatggttaaaatttcaaaaatatgacgagagaaaggttaaacgtacggcatatacttctgaacagagagggaatatggggtttttgttgctcccaacaattcttaagcacgtaaaggaaaaaagcacattatccacattgtacttccagagatatgtacagcattacgtaggtacgttttctttttcttcccggagagatgatacaataatcatgtaaacccagaatttaaaaaatattctttactataaaaattttaattagggaacgtattattttttacatgacaccttttgagaaagagggacttgtaatatgggacaaatgaacaatttctaagaaatgggcatatgactctcagtacaatggaccaaattccctccagtcggcccagcaatacaaagggaaagaaatgagggggcccacaggccacggcccacttttctccgtggtggggagatccagctagaggtccggcccacaagtggcccttgccccgtgggacggtgggattgcagagcgcgtgggcggaaacaacagtttagtaccacctcgctcacgcaacgacgcgaccacttgcttataagctgctgcgctgaggctcaG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

LacZ-OsU3-sgRNA structure in the plasmid

PCR product of LacZ-OsU3-sgRNA (766 bp, 736 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTATGctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcTAAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA (19-20

target

）

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

21

OsU3-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCAAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of OsU3-sgRNA (603 bp, 573 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA(19-20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG

GCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCC ACGCT

LacZ-AtU3b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaatttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

PCR product of LacZ-AtU3b-sgRNA (709 bp, 679 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaatttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcA(19-20 bp target)GTTTTAGAGCTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGT GGATGGNNNNNNNCGAGACCCACGCT

AtU3b-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)

22

PCR product of LacZ-AtU3b-sgRNA (507 bp, 477 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAtttactttaaattttttcttatgcagcctgtgatggataactgaatcaaacaaatggcgtctgggtttaagaagatctgttttggctatgttggacgaaacaagtgaacttttaggatcaacttcagtttatatatggagcttatatcgagcaataagataagtgggctttttatgtaatttaatgggctatcgtccatagattcactaatacccatgcccagtacccatgtatgcgtttcatataagctcctaatttctcccacatcgctcaaatctaaacaaatcttgttgtatatataacactgagggagcaacattggtcA(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

PCR product of LacZ-AtU3d-sgRNA (486 bp, 456 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAATAAAGGAGGCAGCTatgctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggctaaataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtca (19-20 bp target) GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG TGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU3d-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAataagctt atgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcaAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone）

PCR product of AtU3d-sgRNA (284 bp, 254 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAataagcttatgatttcttttttcttacgaattttgcgtcccacatcggtaagcgagtgaagaaataactgctttatatatggctacaaagcaccattggtcA(19-20 bp target)GTTTTAGAGCTA GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGT GGATGGNNNNNNNCGAGACCCACGCT

PCR product of AtU6-1-sgRNA (487 bp, 457 bp after Bsa I digestion)

23

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAGACGAAGTAGTGATTG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCA CCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

AtU6-29-sgRNA structure in the plasmidacccggGGATCCTAGCCGGGTCTCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGAATTTTCCTCCGTTTTACCTGTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattgAGAGACCTCTGAAGATAACATACTAAGCTTggcact(pUC18backbone)PCR product of AtU6-29-sgRNA (502 bp, 472 bp after Bsa I digestion)

TTCAGAGGTCTCTNNNNNNNTGGAATCGGCAGCAAAGGAaaatatcagagatctcttacagttagtttcgttcttaatccaaactactgcagcctgacagacaaatgaggatgcaaacaattttaaagtttatctaacgctagctgttttgtttcttctctctggtgcaccaacgacggcgttttctcaatcataaagaggcttgttttacttaaggccaataatgttgatggatcgaaagaagagggcttttaataaacgagcccgtttaagctgtaaacgatgtcaaaaacatcccacatcgttcagttgaaaatagTagctctgtttatatattggtagagtcgactaagagattG(19-20 bp target)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGNNNNNNNCGAGACCCACGCT

24

本文来源：https://www.bwwdw.com/article/gmep.html