多靶点pCRISPR载体改良版(单子叶植物用)使用方法2014-10-26(P)副本
基因枪轰击法转化单子叶植物
基因枪轰击法转化单子叶植物将愈伤组织高渗处理约4-6h后，用PDS-1000/He 型基因枪进行轰击，每次轰击前，在承载膜上样10ul。
基因枪轰击参数为：氦气压为1100 Psi，真空度为26-27cm 汞柱，承载膜与阻挡屏之间为距离为0.8 cm，可裂膜和承载膜间距离为2.5 cm，愈伤组织轰击距离为9.0 cm。
所有操作过程均在无菌条件下进行, 操作过程如下：（1）用70 %的乙醇对基因枪内外进行消毒，将操做中用到的工具置于超净工作台里，打开紫外灯灭菌30min；（2）用70 %的乙醇将阻挡网，固定器浸泡15 min，对可裂膜用70 %异丙醇浸泡一下，约2- 3 s，微弹载体上用70 %乙醇浸一下放在超净工作台上；（3）打开氦气瓶总阀，并顺时针调节气压，因可裂膜压力为200 psi，调节标准应高于200 psi；（4）打开基因枪面板左侧电源开关，每打完一枪均需将基因枪内所有部位用70 %酒精消过毒的卫生纸擦拭一遍；（5）取微粒载体，，取DNA 与金粉混合物，用加样枪吸取10ul加于承载膜中央，用枪头轻轻将其匀开，防止金粉粒聚集成块状，干燥 2 min，待酒精挥发后，用与基因枪配套的嵌入工具将其固定在钢碗底部环内；（6）安装可裂膜于其托座上，顺时针安装到加速器上，用厂家提供的专用扳手适当用力固定；（7）将空间环，阻挡网，阻挡网托座，承载膜及固定环安装，慢慢旋紧插入枪体中；（8）培养皿根据参数距离放入轰击室，放于中央位置，关好轰击室门；（9）将基因枪上中间气流控制开关置于″VAC″位抽真空，待真空度达到26-27cm 汞柱时，快速按下此开关，按住″HOLD″键，保持不动，直到击发为止，右侧发射开关只有真空压力达到 5 英寸汞柱以上方才点亮。
向上压住发射键″FIRE″并保持不动，轰击结束时，松开″FIRE″键；（10）将气流控制开关置于″VENT″位使其恢复压力值，待真空表到零的位置后将样品取出；（11）关机：把氦气瓶总开关旋紧，打一次空枪，待氦气表指针回零后，逆时针旋转氦气压表调节阀至最小；B 关闭基因枪电源。
CRISPR核酸酶载体试剂盒说明书
CRISPR核酸酶载体试剂盒说明书目录内容和保存介绍产品信息方法实验轮廓设计单链DNA寡核苷酸产生双链寡核苷酸连接反应转化感受态E.coli细胞分析转化子转染哺乳细胞系附录ACRISPR核酸酶载体试剂盒的图谱和特点附属产品技术支持参考附录BCRISPR核酸酶表达细胞的富集内容和含量1.1 双链（ds）对照寡核苷酸序列ds 克隆对照的寡核苷酸的序列如下所列。
ds 克隆对照的寡核苷酸来自退火的，并在试剂盒中提供的50μM的双链寡核苷酸。
ds 克隆对照的寡核苷酸在应用于连接反应之前需要进行再退火和稀释。
5’ CATTTCTCAGTGCTATAGAGTTTT 3’3’GTGGCGTAAAGAGTCACGATATCT 5’1.2感受态细胞转化效率≥1×109 cfu/微克质粒DNA1.3 TOP10细胞的基因型F-MCRAΔ（MRR-hsdRMS-mcrBC）φ80lacZΔM15ΔlacX74recA1 araD139Δ（ARA-LEU）7697Galu galKpsL（STRR）endA1 nupG2 介绍2.1 产品信息2.1.1 介绍GENEART®CRISPR核酸载体试剂盒便于产生表达CRISPR RNA和tracrRNA的非编码RNA以及在哺乳动物细胞中Cas9核酸酶介导的靶基因的裂解或基因编辑的结构体。
该Cas9核酸酶是基于从细菌化脓性链球菌II型CRISPR/ Cas系统，并经过精心设计的基因组编辑在哺乳动物系统（Jinek等人，2012;。
Mali1等人，2013;。
cong等人，2013）。
带有OFP的GENEART®CRISPR核酸载体允许Cas9和CRISPR RNA的基于流式细胞仪表达的细胞群的分选，而带有CD4的GENEART®CRISPR核酸载体使Cas9与CRISPR RNA为基础的表达细胞的富集。
线性GENEART®CRISPR核酸载体提供快速和有效的方式来克隆编码所需CRISPR RNA靶到表达盒，允许Cas9核酸序列中的特定的方式靶向的双链寡核苷酸。
植物CRISPR-Cas9系统高效率基因组编辑
植物CRISPR/Cas9系统高效率基因组编辑ZFN诱导的基因靶向技术早在2003年就已发表。
从那时起，靶向基因组编辑技术得到了迅速发展，并已商业化。
最近，CRISPR/Cas9 通路的发现加快了对该领域的兴趣，为研发开辟了新的可能性。
虽然 CRISPR 通路在细菌中被鉴定为假定的适应性免疫系统的一部分，但它很快适应了修饰真核生物基因组的目的。
虽然像 ZFN 这样的工具为今天的基因组编辑奠定了基础，但这种创始技术和其他类似的技术存在局限性：蛋白质：ZFN的DNA 相互作用使得其设计变得复杂，ZFN 表达构建体的组装非常耗时，并且 ZFN 靶向的选择在许多富含 A-T 的植物基因组中受到限制。
CRISPR通路已经被采用和修改用于真核基因组编辑，克服了许多障碍：它依赖于RNA：DNA相互结合作用以找到其基因组靶标，该结合体的识别序列是易于改变的18-20个碱基对，并且核酸酶结合的唯一要求是在靶位点旁边存在NGG。
CRISPR/Cas9 在研究使用农杆菌瞬时表达分析的植物中的首次于 2013 年报道。
CRISPR/Cas9 技术已成功应用于模式植物（本氏烟草、拟南芥）和作物（水稻、小麦），且名单正在不断增加。
目录：•CRISPR/Cas9：它是什么？它是如何工作的？•利用 CRISPR/Cas9 进行基因组编辑的优势•简化工作流程：植物中的 CRISPR/Cas9•用于单子叶植物和双子叶植物的即用型 Cas9 和向导 RNA (gRNA) 表达质粒•自定义 CRISPR/Cas9 植物产品•自定义 CRISPR/Cas9 订单CRISPR/CAS 9：它是什么？它是如何工作的？CRISPR代表"Clustered Regularly Interspaced Short Palindromic Repeats"（成簇规律间隔短回文重复序列）。
II型原核生物CRISPR“免疫系统”的发现，使得能够开发简单、易用、快速实施的RNA引导的基因组编辑工具。
CRISPR基因编辑技术教程
01
畜禽品种改良
通过CRISPR技术改良畜禽的生长速度 、肉质品质、繁殖性能等性状，培育优 良品种。
02
03
转基因育种
将CRISPR技术与转基因技术相结合， 实现基因的精确编辑和定向转移，加 速育种进程。
04
CRISPR技术挑战与问题
脱靶效应及安全性问题
脱靶效应
CRISPR技术在基因编辑过程中，有时会 出现非特异性切割，导致基因组其他位 置的突变，即脱靶效应。这可能会引发 不可预测的基因功能变化，甚至产生安 全隐患。
通过测序等方法验证表达载体的 正确性和完整性。
转化细胞系或组织
准备细胞系或组织
选择需要编辑的细胞系或组织，并进行适当的预 处理。
转化方法选择
根据细胞类型和组织特性选择合适的转化方法， 如脂质体转染、电穿孔等。
转化效率检测
检测转化效率，确保足够数量的细胞被成功转化 。
验证基因编辑效果
DNA水平验证
通过改进Cas9蛋白的特异性，降低脱靶率，提高基因编辑 的精确度。
开发新型CRISPR系统
探索除CRISPR-Cas9外的其他CRISPR系统，如CRISPRCas12a、CRISPR-Cas13等，以提高编辑效率和特异性。
结合其他基因编辑技术
将CRISPR技术与其他基因编辑技术（如碱基编辑器、先导 编辑器）相结合，实现更高效、更精确的基因编辑。
提取转化后的细胞DNA，通过 PCR、测序等方法检测目标基
因是否被成功编辑。
RNA水平验证
提取转化后的细胞RNA，通过 RT-PCR等方法检测目标基因的 表达水平是否发生变化。
蛋白质水平验证
提取转化后的细胞蛋白质，通 过Western blot等方法检测目 标蛋白质的表达水平是否发生 变化。
精简CRISPR基因编辑技术的方法与应用
精简CRISPR基因编辑技术的方法与应用 CRISPR基因编辑技术是一种革命性的基因编辑工具，它可以高效且准确地修饰生物体的基因组。然而，尽管CRISPR技术在科研界引起了广泛关注，但其复杂性和操作上的难度限制了其在实际应用中的普及。因此，精简CRISPR基因编辑技术的方法和应用的发展显得尤为重要。
精简CRISPR基因编辑技术的方法可以从多个方面进行改进。首先，提高CRISPR系统的效率是关键。目前，CRISPR技术仍存在着目标基因编辑效率不高的问题。一种方法是通过寻找并优化适合目标基因的引物序列，提高获得合适的CRISPR效应子的概率。另外，改进CRISPR基因编辑体系中的Cas蛋白和gRNA的设计也是提高效率的关键。通过对整个CRISPR基因编辑系统的构建进行优化和精简，可以加快基因编辑的速度和准确性。
其次，精简CRISPR基因编辑技术的方法还包括优化基因编辑载体。传统的CRISPR系统通常将Cas蛋白和gRNA构建到不同的质粒上，这样会增加基因编辑复杂度和操作难度。为了精简这一过程，可以采用单一的、高效的载体，将Cas蛋白和gRNA同步表达，从而简化基因编辑的步骤。此外，还可以优化CRISPR载体的传递方式，例如，使用病毒载体可以提高基因编辑的效率和成功率。
精简CRISPR基因编辑技术的方法还包括创新性地应用在不同领域中。CRISPR技术在生物医学研究、人类疾病治疗以及农业生产等众多领域展现了巨大的应用潜力。例如，在生物医学研究中，CRISPR技术被用于研究特定基因缺陷对人体健康的影响，从而开辟了治疗某些遗传性疾病的新途径。另外，CRISPR技术还可以用于改良和提高农作物的耐旱性、抗病性和产量等重要农业性状，以满足全球不断增长的食品需求。
除了基因编辑外，CRISPR技术还可以用于其他方面的应用，如基因调控和基因表达。通过调节CRISPR-Cas9系统的相关组分，即可将其从基因编辑工具转变为基因开关，实现基因的开关式调控。这种方法使得科研人员可以精确操控目标基因在细胞或生物体中的表达水平，从而有助于揭示基因调控网络的复杂性。
改进的CRISPR基因组编辑使用小型高活性和特异性的工程RNA引导核酸酶说明书
Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases Moritz J. Schmidt1, Ashish Gupta1, Christien Bednarski1, Stefanie Gehrig-Giannini1, Florian Richter1, Christian Pitzler1, Michael Gamalinda1, Christina Galonska1, Ryo Takeuchi2, Kui Wang2, Caroline Reiss2, Kerstin Dehne1, Michael J Lukason3, Akiko Noma2, Cindy Park-Windhol2, Mariacarmela Allocca2, Albena Kantardzhieva2, Shailendra Sane2, Karolina Kosakowska2, Brian Cafferty2, Jan Tebbe1, Sarah J Spencer3, Scott Munzer2, Christopher J. Cheng2, Abraham Scaria2, Andrew M. Scharenberg2, André Cohnen1* and Wayne M. Coco1* Supplementary Figure 1. Selection of four small, novel Staphylococcus Cas9s.(a) Relatedness and genomic context of four uncharacterized Staphylococcus Cas9 genes: S. hyicus (Shy), S. lugdunensis (Slu), S. microti (Smi) and S. pasteuri (Spa). Each Cas9 locus includes several direct repeats (DRs) and a tracr sequence upstream of the nuclease. Loci not drawn to scale. (b) Sequences and structure of RNA components. The associated elements shared 98.8 % (tracrs) and 97.2 % (DRs) sequence identity with the corresponding SauCas9 sequences1. Top left: Complementarity between Slu genome locus DRs and tracr sequence. Bottom left: Alignment of the four DR sequences. Blue box: non-identities. Right: sgRNA design for SluCas9 with GAAA tetraloop (Loop 1) fusing DR and tracr2. Secondary structure predictions indicated similar folding for the four tracrRNAs. (c) Alignment of known and putative PAM interacting motifs in the selected Staphylococcus Cas9s suggests PAM-interacting residues. Amino acid numbering based on SauCas9. Strictly conserved amino acids are inblue, chemically similar amino acids are green. Red boxes correspond to residues responsible for PAM recognition in SauCas93. (d) PAM sequences as web logos. PAMs of the Cas9s were identified by in vitro-cleavage assays on a heptanucleotide (N7) DNA library 3’ of VEGFA_T24 and confirmed by bacterial survival assays carrying the same libraries. In contrast to SauCas9, 3 of the 4 chosen Cas9s recognized the short, non-degenerate 5’-NNGG-3’ PAM. PAM numbering begins with the first position after the last 3’ guide nucleotide. Source data are pr ovided in the source d ata file. (e) Alignment to SauCas9 nuclease active site residues. Corresponding amino acids for each orthologue are highlighted with red boxes.Supplementary Figure 2. Genome editing with four Cas9s in HEK293T cells assayed by amplicon sequencing. Shy, Smi and Slu Cas9 activity on endogenous loci in HEK293T cells using SluCas9 tracrRNA. Spa, Smi and Slu were tested for 2 targets with 5’-NNGG-3’ PAM (guide_87 and guide_102 targeting the HBB_R01_T2 and VEGFA_T22 loci, respectively) and Shy with 5’-NNARMM-3’ PAM (guide_1-4 targeting the HBB_R01_T1, VEGFA_T1 and FANCF_T1 and FANCF_T2 loci). Cas9s were delivered as RNPs via nucleofection and editing was analyzed via amplicon sequencing (AmpSeq). For negative controls, the respective nucleases were nucleofected in absence of sgRNA. Editing values were normalized against background, n = 2 independent biological replicates, source data are provided in the source data file.Supplementary Figure 3. Screening assays for functional sRGN variants. (a) Screening approach used to identify improved sRGNs by protein engineering. The succession of screening assays is shown on the left with the corresponding number of sRGN variants that progressed on the right. 25 sRGN variants were identified as top hits in the final BFP disruption screen in HEK293T cells5. (b) Schematic for “live/dead” (L/D) bacterial survival assay. Cells were generated harbouring an arabinose-inducible toxic ccdB gene reporter plasmid, a second plasmid harbouring a transcription cassette for the corresponding sgRNA, and a third plasmid encoding an IPTG-inducible, Trc promot er-controlled nuclease gene. Active Cas9/sgRNA complexes successfully cleave the toxic reporter, which is inactivated by cleavage at the VEGFA-T2 target site (guide_113), and cells survive under selection conditions. Open circles are cartoons of plasmids, green circle depicts ccdB gene product, purple circle represents nuclease. (c) L/D assay validation using SpyCas9 and SpyCas9 mutants. WT = SpyCas9; D10A and H840A = nickases; and dSpyCas9 = catalytically inactive SpyCas9. Upon botharabinose and IPTG induction, SpyCas9-or D10A-expressing cells survive while those that express H840A or dSpyCas9 do not, WTSpyCas9 n = 2 independent biological replicates, all other data n =3 independent biological replicates, data are presented as mean ± SD. Source data are provided in the source data file. (d) Fluorescence polarization assay (FP Assay) schematic. Biotinylated and ATTO647N-labelled oligonucleotide duplexes were immobilized on streptavidin coated plates. RNP complexes were formed, and cleavage of the dsDNA was monitored by following decreasing anisotropy and increasing fluorescence intensity, arb.unit = arbitrary units.Supplementary Figure 4. Rational exchange of PAM-interacting domains. (a) Shuffling fragment architecture of screening hits sRGN1, sRGN2, sRGN3 and sRGN4. Dark blue = SluCas9 segments, light blue = ShyCas9 segments, green = SmiCas9 segments, pink = SpaCas9 segments. (b) Rationally swapping PI-domains (PID) and WEDGE/PI-domains from the NNGG-recognizing SluCas9 to ShyCas9 alters PAM motif recognition and creates functionally active proteins. Chimera 1 and 2 substitute SluCas9 WEDGE/PI domain amino acids 739-1053 and 910-1053, respectively, into the ShyCas9 gene. Bacterial live/dead growth analysis on the VEGFA_T2 target sequence (guide_113) with a NNGG-PAM, indicates that both chimeric constructs gained the ability to effectively employ an NNGG PAM motif, while the unaltered ShyCas9, as expected, cannot. Data presented is the mean of n =2 independent biological replicates, source data are provided in the source data file. PLL = phosphate lock loop, CTD = C-terminal domain, WED = WEDGE domain, TOPO = topoisomerase domain, Ara = arabinose.Supplementary Figure 5. Catalytic turnover and indel patterns. (a) Plasmid containing the on-target sequence, 5'-TCGTAAAGTGGTGCGTTCTC-3', was mixed with the indicated nuclease at a DNA:RNP molar ratio of 2:1. At the indicated times, reactions were quenched and analyzed, data presented are n =1 biological replicate. Stoichiometric product formation ratios greater than 1 indicate that single nuclease molecules cleave d multiple substrate molecules. (b) Insertion and deletion (indel) pattern for SpyCas9, SluCas9 and sRGN3.1 on eight target sites within the VEGFA locus (for guide sequences, see Supplementary Table 1). Amplicon sequencing was performed and indel identity was calculated by CRISPResso. Data are presented as mean of n =2-3 independent biological replicates, except Spy (guide_37) n = 1 independent biological replicate, source data are provided in the source data file. Unaltered reads (indel = 0 bp, black squares) were excluded from the analysis.Supplementary Figure 6. Genome editing in a murine hepatoma cell line. Genome editing performance of SpyCas9, SluCas9, sRGN3.1, sRGN3.3, sRGN1, sRGN2, and sRGN4 variants in murine cells was assessed by transfection of Hepa1-6 cells with the respective albumin locus-targeting mRNAs (see Supplementary Table 2) and sgRNAs (Alb-T1 target, guide 112, see Supplementary Table 1 for sequence), n = 3 independent biological replicates for sRGNs and SluCas9, data are presented as mean ± SD, and n = 2 for SpyCas9). Source data are provided in the source data file.Supplementary Figure 7. Extended activity and specificity assessments. (a) Activity comparison plots of sRGN3.1 vs. SpyCas9 and SluCas9 on 96 targets. 48 therapeutically relevant targets and 48 rationally designed targets (engineered to explore GC-content between 20% and 80 %, see Supplementary Table 1 for sequences) were probed with SpyCas9, SluCas9 and sRGN3.1 in the FP cleavage assay. Each dot depicts the mean initial slope for each reaction, arb.unit = arbitrary units. Line visualizes x = y; data are presented in mean and are n = 2 independent biological replicates. (b) Left: Nuclease specificity assessment by cell-free FP assay at all possible single nucleotide target::gRNA mismatches. Heatmap ranges from white (no off-target cleavage) to dark blue (extensive off-target cleavage), purple box = WT nucleotide. PAM (not depicted) is to the right of the displayed sequences. Middle: alternative depiction of these data with relative initial rate values (slope) for each mismatch position, colors indicate the respective mutation at each position. Overall specificity for SpyCas9 and SluCas9 was about equal, while overall specificity of sRGN3.1 was 15% higher than SpyCas9. Right: on-target cleavage in these experiments relative to SpyCas9, data are presented as mean with n =2 independent biological replicates. (c) On-target editing observed with the nuclease concentrations selected by titration as input for the GUIDE-Seq experiment, with or without double-stranded oligodeoxynucleotide (dsODN), required for capturing of off-targets in GUIDE-Seq experiments. 60 pmol SpyCas9 and 30 pmol each of SluCas9 and sRGN3.1 were used for targeting HBB-R01, except for sRGN3.1 with 22nt guide, 8pmol were used. For targeting VEGFA_T2, 60 pmol SpyCas9, 8 and 18 pmol sRGN3.1 (for 20nt and 22nt guide, respectively) and 6 and 10 pmol SluCas9 (for 20nt and 22nt guide, respectively), n = 1.(d) Quantification of off-targets retrieved for each nuclease by GUIDE-Seq by number of mismatches (left) or overall number of off-targets (right). Results for both targets (HBB_R01 and VEGFA_T2) were combined for these plots. Source data for all subfigures are provided in the source data file.Supplementary Figure 8. LNP-mediated in vivo editing of sRGNs. (a)UPLC analysis of sRGN3.1 and SpyCas9 LNPs. Left: Representative chromatogram. sRGN3.1 mRNA LNPs had a size of 74 nm, polydispersity index (PDI) of 0.11, and RNA entrapment of 96%; SpyCas9 mRNA LNPs had a size of 74 nm, PDI of 0.13, and RNA entrapment of 91%. Lipid standards (right) were analyzed to identify peaks. sRGN3.1 LNPs showed a slight reduction in amino lipid(AL) and an increase in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1(Δ9-Cis)PE (DOPE) phospho-lipid (Phos) content compared to SpyCas9 LNPs, suggesting altered lipid-RNA associations compared to SpyCas9 mRNA, arb.unit = arbitrary units, CH = cholesterol, PEG =poly-ethylene-glycol, black line = background. (b) CryoTEM morphology analysis of sRGN3.3 and SpyCas9 LNPs. sRGN LNPs displayed improved circularity and multilamellar structure. One representative image of preparations shown in 8 c. (c) SpyCas9-LNP and sRGN3.3-LNP functional stability at 4°C. Liver editing in mice was assessed on indicated days upon intravenous administration. Left: dose of 2 mg/kg, n = 4 independent biological replicates, mean ± S.D. Right: Normalization of the data to day 1 showed 3.6-fold activity reduction for SpyCas9-LNPs and 1.3-fold activity reduction for sRGN3.3-LNPs. (d) Functional in vivo evaluation via TIDE analysis of uridine depletion for sRGN3.1 and sRGN3.3 mRNA constructs with different uridine-substituted base modifications. sRGN3.3 mRNA with (N1)-methylpseudouridine (m1Ψ) or sRGN3.1 with pseudouridine (Ψ) showed no significant difference with uridine depletion; whereas sRGN3.1 mRNA with m1Ψ, 5-methoxyuridine (5moU), and no modification showed significantly increased editing with uridine depletion, (dose of 1 mg/kg, n = 4 independent biological replicates, mean ± SD). For all other in vivo studies m1Ψ modification and the non-uridine depleted constructs were used. Significance was determined using the Mann Whitney test, (*) = p < 0.05, ns = not significant. (e) In vivo evaluation of sgRNA modification approaches in mice via TIDE analysis. Tested were internal chemical modifications (Internal mod 1 and 2) and increasing protospacer length from 20 to 23 nt (Supplementary Table 1). Liver editing showed a dose response at 0.5, 0.75, and 1.5 mg/kg of total LNP-encapsulated RNA. Both modification strategies showed improved potency compared to standard modified sgRNAs. Protospacer length of 23 nt (standard modifications) showed highest potency. N = 4 independent biological replicates, mean ± SD. Source data are provided in the source data file.Supplementary Figure 9. Editing at the intron 40 of the USH2A gene locus. (a) The 7595-2144A > G Usher disease mutation in intron 40 of USH2A (IVS40) results in an additional exon being incorporated in the USH2A mRNA. The T429 IVS50 guide (guide_110) targets the mutant IVS40 allele, while WT-IVS40 targets the WT (Supplementary Table 1, guide_111). GUIDE-seq revealed no sRGN off-target sites above background for T428-IVS40-guide and confirmed via AmpSeq (source data file, Supplementary Fig. 9). A plasmid carrying the sRGN3.1 gene and T428-IVS40-guide was nucleofect ed into the homozygous IVS40 293FT (293FT-IVS40) cell line together with dsODN. Break sites in which dsODN was inserted were identified by NGS. Only genomic sequences quite distant (> 7 mismatches and multiple alignment gaps) from the on-target site for T428-IVS40-guide were captured by dsODN at relevant read counts, suggesting that these sites were not cleaved as off-targets by sRGN3.1 complexed with T428-IVS40-guide. Background was 0.26 % for SpyCas9 and 0.07 % for sRGN3.1. Three replicates for sRGN3.1are shown, identical scales for each replicate. (b) Comparison of editing with mutant IVS40 allel e targeting guide and its surrogate guide. 293FT-IVS40 cell line and its WT parent cell line were transfected with a plasmid carrying either SpyCas9 or sRGN3.1 and sgRNA that matched either WT (guide_111) or the IVS40 SNP allele(guide_110). WT-IVS40-guide differs from T428-IVS40-guide by a single nucleotide and completely matches the wild type USH2A intronic sequence of NHP and human. Insertions and deletions (indels) were quantified using cells harvested 7 days after transfection via TIDE analysis, n = 2 biologically independent experiments, 2 technical replicas each, each datapoint is shown. Source data are provided in the source data file.Supplementary Figure 10. Gating strategy for FACS analysis of BFP-disruption. HEK293T cells, harboring a BFP cassette in the AAVS1 locus were transfected with nuclease expression plasmid (with T2A-GFP) and guide expression plasmid (guide 5-9 and 11-16 for sRGNs and guide_19-23and 25-30 for SpyCas9). Cells were FACS-analyzed 7 days post transfection. Shown is the gating strategy for the control (nuclease only) and a nuclease plus guide treated sample, as used for evaluation of % BFP disruption as presented in Figure 2. A signal from minimum of 10,000 cells was assayed using the V450 filter set in the BD FACS canto II and software diva 8.0.1 and FlowJo 10.7.2.Supplementary References1. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature520,186–191 (2015).2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptivebacterial immunity. Science337, 816–821 (2012).3. Nishimasu, H. et al. Crystal Structure of Staphylococcus aureus Cas9. Cell162, 1113–1126 (2015).4. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases inhuman cells. Nature Biotechnology31, 822–826 (2013).5. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAMspecificities. Nature523, 481–485 (2015).。
手把手教你学会CRISPRCas9基因敲除技术（需要挑选成对的靶点一般在正义链和反义链上分。。。
⼿把⼿教你学会CRISPRCas9基因敲除技术（需要挑选成对的靶点⼀般在正义链和反义链上分。
h ttp:///a/214091994_177233(需要挑选成对的靶点⼀般在正义链和反义链上分别挑选靶点配对)C RISPR/Cas 是进⾏基因编辑的强⼤⼯具，可以对基因进⾏定点的精确编辑。
在向导 RNA（guide RNA， gRNA）和 Cas9 蛋⽩的参与下，待编辑的细胞基因组 DNA 将被看作病毒或外源 DNA，被精确剪切。
⼀、寻找⽬的基因的靶标使⽤在线设计⽹站 CRISPR direct，如需直接复制⽹址，可在⽣物学霸后台对话框回复 direct即可。
靶点挑选要点：1. 基因敲除靶点应设计在起始密码⼦附近（包括起始密码⼦）或者起始密码⼦下游的外显⼦范围内。
2. 不同Cas9/gRNA 靶点在基因敲除效率上有较⼤差异，因此同时设计构建2～3 个靶点的基因敲除载体再从中选出敲减效果较佳的靶点。
3. N1-N20NGG 靠近 PAM 的碱基对靶点的特异性很重要，前 7～12 个碱基的错配对 Cas9 切割效率影响较⼩。
设计好的靶点序列应在基因库中进⾏ BLAST 检测。
4. Cas9Nicknase 需要挑选成对的靶点。
⼀般在正义链和反义链上分别挑选相距 20～30bp 的靶点配对。
多对靶点的敲除效率常有较⼤差异。
由于基因敲除实验时间长，在正式对⽬的细胞进⾏敲除前对靶点进⾏验证和挑选⾮常必要。
⼆、插⼊⽚段设计插⼊寡核苷酸序列设计（必须 PAGE 纯化寡核苷酸）：正向序列5’ACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT3’反向序列3’TGTGGCNNNNNNNNNNNNNNNNNNNCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAA5’插⼊⽚段的合成1. ⽤⽔将寡核苷酸稀释为 100 µM。
pSPYCE(MR)植物表达载体
pSPYCE(MR)编号 载体名称北京华越洋生物VECT0380 pSPYCE(MR)pSPYCE(MR)载体基本信息出品公司: -‐-‐载体名称: pSPYCE(MR)质粒类型: 植物双元表达载体高拷贝/低拷贝: -‐-‐启动子: -‐-‐克隆方法: 多克隆位点，限制性内切酶载体大小: -‐-‐ 5' 测序引物及序列: -‐-‐ 3' 测序引物及序列: -‐-‐ 载体标签: -‐-‐ 载体抗性: -‐-‐ 筛选标记: -‐-‐ 备注: -‐-‐ 产品目录号: -‐-‐ 稳定性: -‐-‐ 组成型: -‐-‐ 病毒/非病毒: -‐-‐pSPYCE(MR)载体简介其他植物载体质粒：pBI101 pDF15pBI121 pEarleyGate 100pBI221 pEarleyGate 101pBI221-‐GFP pEarleyGate 102pBin19 pEarleyGate 103pBINPLUS pEarleyGate 104pCambia0105.1R pEarleyGate 201pCambia0305.1 pEarleyGate 202pCambia0305.2 pEarleyGate 203pCambia0380 pEarleyGate 204pCambia0390 pEarleyGate 205pCambia1105.1 pEarleyGate 301pCambia1105.1R pEarleyGate 302pCambia1200 pEarleyGate 303pCambia1201 pEarleyGate 304pCambia1281Z pFGC5941pCambia1291Z pGA643pCambia1300 pGreen pCambia1300GFP pGreen 0029 pCambia1301 pGreen0029 pCambia1302 pGreen029 pCambia1303 pGreenII pCambia1304 pGreenII 0049 pCambia1305.1 pGreenII 0179 pCambia1305.2 pGreenII 0229 pCambia1380 pGreenII 0579 pCambia1381 pHANNIBAL pCambia1381Xa pHELLSGATE pCambia1381Xb pHELLSGATE 12 pCambia1381Xc pHELLSGATE 4 pCambia1381Z pHELLSGATE 8 pCambia1390 pKANNIBAL pCambia1391 pPZP100 pCambia1391Xa pPZP101 pCambia1391Xb pPZP102 pCambia1391Xc pPZP111 pCambia1391Z pPZP112 pCambia2200 pPZP121 pCambia2201 pPZP122 pCambia2300 pPZP200 pCambia2301 pPZP201 pCambia2301-‐101 pPZP202 pCambia3200 pPZP211 pCambia3201 pPZP212 pCambia3300 pPZP221 pCambia3301 pPZP222 pCambia35s-‐ECFP pPZp-‐RCS2-‐Bar pCambia35s-‐EGFP pRI 101-‐AN pCambia35s-‐EYFP pRI 101-‐ON pCambia5105 pRI 201-‐AN pSB1 pRI 201-‐ON pSB11 pRI 909 pSoup pRI 910 pSPYCE(MR) pRI101pTCK303 pSAT1-‐cCFP-‐C Super1300 pSAT1-‐cCFP-‐N pSAT6nCeruleanC(A+) pSAT4-‐nVenus-‐C。
CRISPR-Cas系统与植物基因编辑
马铃薯
CRISPR/Cas技术在单子叶植物中的应用
CRISPR-Cas9基因编辑技术
水稻
小麦
玉米 高粱
矮牵牛
水稻
水稻是人类的主要粮食作物，选 育优良和高产量的品种有助于解决世 界上人口与粮食不足的问题. 而且水 稻也是单子叶植物中的一种模式植物。 2017年，沈春修靶向水稻的LOC_Os 10g05490基因，将构建的重组质粒 利用电激法转化到农杆菌中，并转化 水稻胚性愈伤组织，最后获得高达76. 5%的转化率。
烟草
CRISPR-Cas9基因编辑技术
大豆是重要的粮食作物. 2015年Su n等针对大豆的Gm06g14180、Gm08 g02290和Gm12g37050基因设计靶 位点，分别用拟南芥的AtU6启动子和 大豆的GmU6启动子驱动gRNA，获得 了3.2%-20.2%的突变率且使用大豆G mU6启动子比拟南芥AtU6启动子的打 靶效率更高；其内源的启动子也可以 驱动gRNA实现定点突变。结果证明C RISPR/Cas9系统可以对大豆的外源导 入基因及内源基因进行定点基因编辑。
CRISPR-Cas9基因编辑技术
03
四种不同介导的 植物基因编辑技术
四种介导因编辑技术
HR介导的基因 定点插入或替换
CRISPR-Cas9系统 介导的基因 激活和干扰
NHEJ介导的基因敲除
CRISPR-Cas9基因编辑技术
实际应用中， 有些植物的性状通常由多个基因控制， 而传统的获得多基因突变 体的方法费时费力， ZFN和TALEN技术在靶点设计上的繁琐也限制了其同时敲除多 基因的功能。因此， 利用CRISPR-Cas9技术对多基因的敲除更显其优越性。多基因 的敲除的核心问题在于多靶点， 因此只要解决了多个sgRNA表达框的问题就可将问 题简化。
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CRISPR/Cas9-based genome editing technologyA CRISPR/Cas9 vector system for multiplex targeting of genesites in monocot 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（**************.cn ）1. CRISPR/Cas9核酸酶切靶序列原理图5’ NNNNNNNNNNNN GNNNNNNNNNNNNNNNNNNNTarget SitePAMNNNNNNNNNNNN-3’3’ NNNNNNNNNNNNNCCNNNNNNNNNNNN-5’NNNNNNNNNNNNNNNNNNNN5’-G/A NNNNNNNNNNNNNNNNNNNN GUUUUAGAGCUAGA ACGAUA GAAAACUAUUGCCUGAUCGGAAUAAAAUUCas9 nucleaseCleavage sitegenomesequenceRuvC-like domain HNH domainCUUGAAAAAGUGGCACCGAGCGUGGCUUUUUU-3’NGG2 相关载体图谱2.1 CRISPR/gRNA vectors (单子叶植物用) (sgRNA: single guide RNA,简称gRNA)：注：用载体骨架长片段隔开U3/U6启动子和gRNA 区可避免未切断质粒被PCR 扩增（短时延伸20秒)见后面）。
这些质粒在E.coli DH10B 繁殖。
2.2 CRISPR/Cas9双元载体(单子叶植物用)pYLCRISPR/Cas9-MH ，原命名pYLCRISPR/Cas9-MTmono (M=monocot; H=HPT ，抗潮霉素基因)pYLCRISPR/Cas9-MB (B=Bar, 抗草苷膦基因)本套载体系统的pCas9为本实验室设计合成的植物优化密码子基因。
双元载体骨架（LB —RB 非T-DNA 序列）为pCAMBIA-1300(ACCESSION: AF234296)与以前的载体相比，这2个载体改造了原来的融合型ccdB 即LacZ/ccdB 为ccdBs (shortenccdB ,即删除了LacZ 序列)及其相对方向反过来，其它部分不变。
pYLgRNA-OsU6a~c ; -OsU3Bsa I (1)Bsa I (2)Hin dIII Bam HIOsU3, OsU6a,b,cpUC18 backboneAmp rBsa IgRNApYLgRNA-OsU3/LacZBsa I (1)Bsa I (2)Hin dIIIBam HIU3/6upstream primersgRNA down stream primers OsU3pUC18 backboneAmp rBsa IgRNAE.coli PrLacZa gagacc TCTGA----GCGTC ggtctc a GTTT Bsa I (1)Bsa I (2)pUC18 backboneOsU6a GGC A OsU3GCC G OsU6b GTT G OsU6cTCA GRice small nuclear RNA promotersBsa I cutting :NNN ggtctc NNNNNNN-33-NNN ccagag NNNNNNNgRNAt ctctgg AGACT----CGCAG ccagag a CAAALacZ Mlu I gRNA down stream primersU3/6upstream primers注： 这些质粒保存在 E. coli TOP10F ’(LacI q ) 菌株繁殖，以使ccdB(大肠杆菌致死基因)能够被LacI q 产生高水平阻碍蛋白抑制其表达。
3. 基因组靶位点选择和双链接头设计 3.1 靶位点选择（1）在目标区如果能够找到NGG 上游第20碱基是A （用U3启动子）或G (用U6a~c 启动子）的序列 (A ，G 分别为U3和U6启动子的转录起始碱基），优先选为靶序列GCGCGGTGTCA TCTATGTTACTAGATCGGGAGCACCGGTAA GG CGCGCC GTAGTG CTCG A GAGACC TCTG AAG(ccdBs,657bp )GAGCGTG GGTCTC G CGGT ATCATT GGCGCG CC TCTCGAGCTA GCGGCCGC ATGC ATCGATCTC CTACATCGTATAAATTAGCCTATACGAAGTTATTGCATCTATGTCGGGPCR/Sequencing primer (SP-R, 原命名SP1):PCR/Sequencing primer (SP-ML, 原命名SP2)Asc I B-L Bsa IBsa I B-R Asc I Not ICCCGACATAGATGCAATAACTTC-3I(Nuclear Localization Signal)I5-NNNNNNNNN A NNNNNNNNNNNNNNNNNNN NGG 靶位点G 4碱基酶切位点，有利于检测打靶效果合成接头的形式（左：连接U3启动子；右：连接U6a 启动子）注意：接头正向引物F 和反向R 命名命名是根据靶点在gRNA 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） 如果在NGG 上游第20碱基不是A 或G ，可选20碱基为靶序列（参考Kabin Xie and Yinong Yang ， 2013， Molecular Plant)合成接头的形式（连接U6a 启动子）也就是说，所用启动子的转录起始点与NGG 上游第20碱基相同的靶点就是19碱基(19+A/G=20)，不相同的靶点就是20碱基。
（3）为了提高突变效率，可以对一个目的基因设计2个靶点(尤其只有一个靶基因)。
建议在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。
靶点序列GC%偏高可提高打靶效率，因此靶点最好含有11-14个C/G （包括U6转录起始点G ）。
几个靶位点设计例：左图：切点在起始密码附近或尽量在ORF 上游，或在特定的功能域 (可能引起密码子缺失和移码)右图：切点在外显子末端或前端（可能引起移码，或内含子识别位点缺失使内含子不被剪切）（4）靶点特异性检查：虽然对植物基因打靶的特异性不是重要的问题（万一产生有负面作用的非特异打靶，把突变体与原受体亲本杂交（和回交）就可分离排除非特异打靶位点），但应该将候选靶序列+NGG （上下游加几十碱基）对目标基因组做Blast (选Somewhat similar sequences (blastn)，避免在靶序列3’端+NGG 与其它功能基因和基因组序列有相似性。
但是打算用一个靶序列敲除2个或以上的同源基5-ggc A NNNNNNNNNNNNNNNNNNN -3’3-NNNNNNNNNNNNNNNNNNN CAAA-5’19nt 接头正向引物反向引物5-gcc G NNNNNNNNNNNNNNNNNNN -3’3-NNNNNNNNNNNNNNNNNNN CAAA-5’19nt5-gcc G NNNNNNNNNNNNNNNNNNNN -33-NNNNNNNNNNNNNNNNNNNN CAAA-520ntGG -3起始密码PAMG T ATGATAATGCAGTATGGTTA-3内含子外显子5-NNNNNNNNN NNNNNNNNNNNNNNNNNNNN NGG NNNNNNNNN-3PAM20nt因时，就选择几个目标基因完全相同的区域为靶位点。
3.2 靶位点接头引物设计例4. 多靶点pYLCRISPR/Cas9-MH (B)载体构建4.1 gRNA 表达盒构建示意图4.2 靶点引物接头与gRNA 表达盒的实际连接方式和扩增为了提高接头连接效率，使用较高浓度（0.05-0.1 M ）的靶点接头，实际上大部分产生线性单端连接，而环状(双端)连接较少。
另外，过度酶切，星活性，过度连接等可能产生破坏的平滑末端，有可能连接产生双接头产物或的空载产物（下图的产物IV 和产物V ）。
连接接头后，有3种方法扩增gRNA 表达盒片段：（1）直接用位置特异引物做一轮PCR 扩增; （2）做2轮巢式PCR ，第一轮PCR 用通用的U-F/gRNA-R 做1个反应，第二轮用位置特异引物扩增；（3）做2轮巢式PCR ，第一轮PCR 做2个反应，3’3’accattcacgggggtagga g -5’20bpggc Ag aggatgggggcacttacca ctcctacccccgtgaatggt caaa -5’-3’5’-3’-For U3-gRNAgcc G aggatgggggcacttacca tcctacccccgtgaatggt caaa -5’-3’5’-3’--For U6a-gRNA gtt G aggatgggggcacttacca tcctacccccgtgaatggt caaa -5’-3’5’-3’--For U6b-gRNAtca G aggatgggggcacttacca tcctacccccgtgaatggt caaa -5’-3’5’-3’--For U6c-gRNA20bp19bp分别用U-F/接头反向引物，和用接头正向引物/gRNA-R ；第二轮为Overlapping PCR,用位置特异引物扩增出表达盒产物。
方法（1）效果不那么稳定，且不能避免扩增下图的产物IV 和产物V 。
方法（2）的扩增效果好且稳定，但同样不能避免扩增产物IV 和产物V 。
方法（3）虽然第一轮PCR 需要做2个反应，但效果最好且能避免扩增产物IV 和产物V ，因此推荐使用方法（3）。
下图为方法（3）示意图。
在第一轮PCR 加热过程中（73-75度），有缺口的引物（Tm 值低于U3/6和gRNA 序列）先解离， U3/6和gRNA 序列3’端（未解离）立即延伸补平，产生接头引物互补结合位点。
4.3 gRNA 表达盒扩增引物第一轮PCR 扩增引物（所有gRNA 表达盒共用）：U-F: 5-CTCCGTTTTACCTGTGGAATCG-3; gRNA-R: 5-CGGAGGAAAATTCCATCCAC-3第二轮PCR 扩增引物（使用策略I Golden Gate ligation 的位置特异gRNA 表达盒专用）：Two Bsa I-cut ends are linked to different adapterU3(6)upstream primer U-F /Reverse adapter primerFirst PCR (2reactions):Reaction 1and the product V is not amplified.Two Bsa I-cut endsare linked to an adapterTwo adapter ends of product s II & III are polished to blunt and linked to produce IVTwo Bsa I-cut ends are polished to blunt and linked togetherForward adapter primer/gRNAdownstream primerReaction 2II IIIIV V（B1’, B2, B2’, B3, B3’等表示各连接点位置；ctcg, tcag等表示Bsa I切点粘性末端的正链序列;相同颜色的Bsa I切点粘性末端是可特异配对连接的互补末端）靶点1（T1）：SpeI（new）Uctcg-B1’: T TCAGA ggtctc T ctcg ACTA GT GGAATCGGCAGCAAAGG-3（U3/6上游引物）gRctga-B2: AGCGTG ggtctc G tcag GG TCCATCCACTCCAAGCTC-3（第二轮gRNA下游引物）T2:Uctga-B2’: T TCAGA ggtctc T ctga CAC TGGAATCGGCAGCAAAGG-3gRaaga-B3: AGCGTG ggtctc G tctt GG TCCATCCACTCCAAGCTC-3T3:Uaaga-B3’: T TCAGA ggtctc T aaga CAC TGGAATCGGCAGCAAAGG-3gRgact-B4: AGCGTG ggtctc G agtc GG TCCATCCACTCCAAGCTC-3T4:Ugact-B4’: T TCAGA ggtctc T gact CAC TGGAATCGGCAGCAAAGG-3gRggac-B5: AGCGTG ggtctc G gtcc GG TCCATCCACTCCAAGCTC-3T5:Uggac-B5’: T TCAGA ggtctc T ggac CAC TGGAATCGGCAGCAAAGG-3gRtctg-B6: AGCGTG ggtctc G caga GG TCCATCCACTCCAAGCTC-3T6:Utctg-B6’: T TCAGA ggtctc T tctg CAC TGGAATCGGCAGCAAAGG-3gRaggt-B7: AGCGTG ggtctc G acct GG TCCATCCACTCCAAGCTC-3T7:Uaggt-B7’: T TCAGA ggtctc T aggt CAC TGGAATCGGCAGCAAAGG-3gRcgct-B8: AGCGTG ggtctc G agcg GG TCCATCCACTCCAAGCTC-3T8:Ucgct-B8’: T TCAGA ggtctc T cgct CAC TGGAATCGGCAGCAAAGG-3(如果要多于8个靶点，通过设计不同的BsaI切点碱基，合成新引物对）最后靶点(Last site) Mlu I（new）gRcggt-BL:AGCGTG ggtctc G accg ACGCG T CCATCCACTCCAAGCTC-3 （与载体RB侧B-R互补）当靶点数少于8个时，gRcggt-BL（BL）作为最后一个gRNA表达盒下游引物替代B2,B3, B4...，见以下引物使用方法)4.4 靶标gRNA表达盒排列和扩增引物使用方法本系统目前使用水稻来源的4个small nuclear RNA启动子（OsU3, OsU6a~c）。