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n. [生]核内小RNA
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...as系统也是目前最常用于人工基因组编辑的CRISPR/Cas系统，其靶向基因组编辑的步骤如图13所示。 图13 利用一段小向导RNA（sgRNA）:Cas复合体系统靶向基因组编辑的步骤。
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对CRISPR/Cas系统中的sgRNA结构进行了优化，以提高CRISPR-Cas9的基因敲除效率。 单导向RNA（sgRNA）是CRISPR-Cas9基因编辑系统两个关键组分的其中一个。
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... 学术报告简介：基因编辑技术的飞速发展，特别是近年来CRISPR技术的广泛应用，使得人类拥有了前所未有的改变和修饰基因组的能力。这项技术利用单链引导RNA(sgRNA)和Cas9蛋白，可以在体内和体外简单、迅速、低成本实现基因编辑。
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...起允许该第一CRISPR酶融合构建体和该第二CRISPR酶融合构建体构成功能性CRISPR-Cas系统，其中该CRISPR-Cas系统包含指导RNA(sgRNA)，该指导RNA包含能够杂交到细胞中的感兴趣的基因组座位中的靶序列上的指导序列，并且其中该功能性CRISPR-Cas系统结合至该靶序列，...
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sgrna 设计方法 sgrna(small guide rna 简称) ,是 ......
通过将表达 sgrna 的原件与表达 cas9 的原件相连接,得到可以同时表达两......
三种方案可选:a, 直接使用pcr扩增纯化产物, 该产物片段含有u6+sgrna,约370 b, 将sgrna载体构建到 pspcas9(bb)-2a-gfppuc19质粒当中,即单载体系统; c, .........
crrsprcas9 基因敲除系统之 sgrna 设计 关键词: sgrna 基因敲除蛋白
00:00 来源:百奥迈科生物 点击次 数:6694 sgrna 设计网站介绍 crrsprcas9 .........
这个 sgrna 足以帮助 cas9 内切酶 对 dna 进行定点切割。 最新的......
关键步骤:hdr 的应用,我们推荐 cloning sgrna guides into one of the sgrna expression plasmids (step 5b) ,而不是用以 pcr 为基础的表达方法。 5 分离.........
在基因组编辑过程中, tracrrna 和 crrna 可以融合成为 1 条 rna(sgrna)表达同样可以起到靶向剪切的作用。 通过基因工程手段对crrna 和tracrrna进行改造,将其.........
ii 型系统中的这两个 rnas 可 以融合成一个 sgrna,目前,ii 型系......
研究者根据果蝇x染色体上红眼基因b设计出sgrna,导入果蝇早期胚胎细_高考_高......
crisprcas9 技术的发展 利用 crisprcas9 技术,通过设计特异性的 sgrna,可以实现 特异性的 dna 识别,并且在靶向位置完成切割,再通过细胞本身的 dna 修复机制,.........
两个小组对天然 crispr-cas 系统进行适当改造, 将 tracrrna 和 crrna 双组分利用基因工程整合为一条链,称为单链引导 rna(single guide rna, sgrna),该 rna .........
载体图谱如下: 二、实验目的 构建带有 pes1 基因 sgrna 和 cas9 基因的表达质粒。 三、 实验步骤 根据人的 pes1 基因设计 sgrna 序列, 安排引物合成。 将单.........
双子叶 植物抗性 潮霉素 2 crisprsgrna vectors sgrna 序列全长 96 nt,分为两部分,5’决定靶序列的 20 碱基 (seed sequence)和 3’区域为保守的结构序 .........
(1)由实验结果分析,sgrna 的导入___了果蝇胚胎的存活率,若要从相同 数量的早期胚胎中获得数量最多的镶嵌体果蝇, 应选用的 sgrna 最佳浓度为___ngml。 (2).........
位于 sgrna5?端的 20 个左右的碱基决定 crisprcas9 系统在......
该过程中, sgrna 和 cas9 酶共同剪切 dna 的作用类似于___酶的......
sgrna 浓度(ngml) 胚胎存活率(%) 镶嵌体果蝇比例(%) 不注射 16 0 0 11 0 125 8 10 250 6 17 500 7 50
(1)由实验结果分析,sgrna 的.........
sgrna 浓度(ngml) 胚胎存活率(%) 镶嵌体果蝇比例(%) 不注射 16 0 0 11 0 125 8 10 250 6 17 500 7 50
(1)由实验结果分析,sgrna 的.........
相较于 cas9-sgrna,ngago-gdna 具有更大的优势,规避了令人头......
我们的一站式服务包括:sgrna 设计、筛选高效的 sgrna、敲除载体构建、敲......
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distribution of the sgrna reads是什么意思
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istribution of the sgrna reads参与分配的读取 重点词汇distribution分配，分布; 配电reads读; 财产分配; 频率分布
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CRISPR-CAS9
Targeted mutagenesis in soybean using the CRISPR-Cas9 systemZheng Hu1,*,#, Xianjun Sun1,2,#, Rui Chen3 , Qiyang Jiang1 , Guohua Song1 , Hui Zhang1 and Yajun Xi2,* 1 National Key Facilities for Crop Genetic Resources and Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China 2 College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China 3 Tianjin Institute of Agricultural Quality Standard and Testing Technology, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China *Authors for correspondence. ZH: ; YX: . # These authors contributed equally to this work.AbstractGenome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce syntheticguide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180,Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2C9.7% using the pCas9-AtU6-sgRNA vector and 14.7C 20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.IntroductionGenome editing is an important tool for gene function analysis, gene therapy and crop improvement.1. zinc-finger nucleases (ZFNs) 2. transcription activator-like nucleases (TALENs) 3. clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system All three methods induce double-stranded breaks (DSBs) in the target genome DNA, which are subsequently repaired through non-homologous end joining (NHEJ) and homologous recombination (HR) 1,2 target the genome through protein-DNA interactions 3 is based on short RNA-DNA base pairing.(simpler, faster and more efficient)The CRISPR-Cas system, an adaptive immunity system against foreign nucleic acid invaders in prokaryotes, exists in most archaea and numerous bacteria. Types I and III contain multiple CAS proteins that form a complex for degrading foreign DNA/RNA. Type II directs the cleavage of targeted foreign DNA using a single CAS9 protein, which makes it the system of choice for targeted genome engineering. Type II ，CRISPR RNA (crRNA) hybridizes with a small trans-activating CRISPR RNA (trancrRNA) to form mature dual crRNA. The mature crRNA combines with Cas9 to form a functional complex. When the complex recognizes a short seed sequence in the vicinity of a typical 5’-NGG-3’ protospacer-adjacent motif (PAM) by RNA/DNA base pairing, Cas9 cleaves the target DNA. Mature crRNA containing trancrRNA and crRNA can be replaced in the laboratory with a single synthetic guide RNA (sgRNA)Cas9 is expressed by the Cauliflower mosaic virus (CaMV) 35s promoter or a gene-specific promoter. A nuclear localization signal (NLS) sequence is fused to the Cas9 gene, which delivers CAS9 to the genomic nuclei. To design the sgRNA, the 20-bp sequence following the PAM in the target DNA is selected as the sgRNA seed. These 20-bp sgRNA seed regions are abundant in plant genomes, with more than 90% of rice genes containing a specific sgRNA seed. Several software programs have been developed to identify target-specific sgRNA seeds. RNA polymerase-III promoters such as U6 and U3 are typically used to express the sgRNA. Several different sgRNAs can be co-expressed within a single CRISPRCas9 system to target multiple DNA sites simultaneously. The CRISPR-Cas9 system is a powerful tool for the advancement of function genomics research. In this study, we used the CRISPR-Cas9 system to efficiently perform targeted mutagenesis in soybean protoplasts and hairy roots. Targeted mutagenesis using the CRISPR-Cas9 system can advance soybean functional genomic research, especially that of genes involved in roots and nodulesMethods? Plant materialThe soybean cultivar Williams 82 was used in this study.? Vector construction? Protoplast isolation and transformation? Transformation mediated by A. rhizogenes? Detection of mutations in target genes? Bioinformatic analysisResultsPrediction of U6 promoters in soybeanThe U6 promoter is typically used to drive the expression of sgRNA in various plantsFigure 1.Alignment of 11 soybean U6 and Arabidopsis U6-26 genes. Soybean U6 and Arabidopsis U6-26 genes are highly conserved. Upstream sequence element (USE), TATA-box and U6 small nuclear RNA (snRNA) sequence regions are underlined.The presence of these conserved elements suggests that the 10 soybean U6 promoters may have the transcriptional activity to generate sgRNAs in soybean.Evaluation of the CRISPR-Cas9 system for gene editing in soybeanWe constructed two binary vectors to express sgRNAs and Cas9 for gene editing (Fig. 2). In both vectors, the CaMV 35s promoter was used to drive the expression of Cas9. Two RNA polymerase-III (Pol III) promoters, AtU6-26 and GmU6-10, were selected to generate sgRNAs in the two vectorsFigure 2.Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. (a) Arabidopsis thaliana U6-26 promoter (b) Glycine max U6-10 promoter. Sequences containing two BsaI sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: RB: right border.To detect the activity of these two vectors in soybean, we selected three genes (Glyma06g14180, Glyma08g02290 and Glyma12g37050) as targets for gene editing in soybean. For each gene, we designed a different sgRNA seed with a restriction site in the vicinity of the PAMTargeted mutagenesis in soybean protoplastsA restriction enzyme PCR (RE-PCR) assay was used to detect mutations in the targeted genes. The mutant genes were not digested as they lost the enzyme sites and could be amplified using the gene-specific primers.Figure 3.Targeted mutagenesis in soybean protoplasts.nucleotide substitutions had occurred in Glyma06g14180 and Glyma12g37050(Figs. 3b,c), suggesting that the DSBs in these two genes were repaired by the HR pathway in the soybean protoplasts. One nucleotide deletion and one substitution were found in Glyma08g02290 (Figs. 3b,c). The DSBs of Glyma08g02290 were repaired through both the HR and NHEJ pathways in soybean protoplastsTargeted mutagenesis in soybean hairy rootsSoybean is a diploid plant and genes have two copies in the homologous chromosomes. The target gene induced by the CRISPR-Cas9 system has three types in the hairy roots. Type I is no mutation of the target gene. Type II is a monoallelic mutation where one gene is mutated and the other allelic gene is no mutated. Type III is a biallelic mutation where both of the two allelic genes are mutatedFigure 4.Detection of mutations using the PCR-restriction enzyme (PCR-RE) assay. (a) Targeted mutations induced by the pCas9-AtU6-sgRNA vector.(b) Targeted mutations induced by the pCas9-GmU6-sgRNA vector.Biallelic mutations can be detected in T0 transgenic plants using the CRISPRCas9 system. We detected several biallelic mutations of Glyma06g14180 and Glyma08g02290 using the PCR-RE assay . A higher frequency of biallelic mutants was observed in Glyma08g02290. 12 of 19 Glyma08g02290 mutants generated using the pCas9-GmU6-sgRNA vector and 2 of 3 Glyma08g02290 mutants induced by the pCas9-AtU6-sgRNA vector were biallelic (Table 1).Figure 5. Gene sequences from 9 independent biallelic mutants.Sequencing of several gene clones from independent biallelic mutant roots revealed a variety of mutations per root , which suggests that the CRISPR-Cas9 system continued to modify the genes during hairy root development.Off-target activity in soybeanThe CRISPR-Cas9 system can tolerate several mismatches between the sgRNA seed and its target, especially in the first 12 nucleotides at the 5’ end of the sgRNA, which suggests that off-target activity is common with the CRISPRCas9 system. We accordingly searched the soybean genome for homologs of the three targeted genes in this study. We found that Glyma06g14180 and Glyma04g40610 had the same target sequence and that the sequences of Glyma08g02290 and Glyma05g37270 were also identical to one another. Glyma12g37050 and Glyma09g00490 differed by a single nucleotide at the PAM site (AGG vs. ATG). Mutations in Glyma04g40610 and Glyma09g00490induced by the CRISPR-CAS9 system using primers for Glyma06g14180 andGlyma12g37050 were detected in protoplasts and hairy rootsDiscussionIn this study, we used two U6 promoters, Arabidopsis U6-26 and soybeanU6-10, to generate sgRNA. Mutation efficiencies in the three target geneswere significantly increased by the use of the soybean U6-10 promoter (Table 1), which may be related to the U6 promoter activity. The transcriptional efficiency of the different U6 promoters varies in Arabidopsis. Eleven U6 promoters were predicted in soybean, which provided the opportunity to select a suitable U6 promoter for the expression of sgRNA in soybean. The choice of promoter is critical, as high concentrations of the Cas9-sgRNA complex can increase off-target activity.The mutations induced by T-DNA insertion, chemical agents and physical treatments are random, which make it difficult to obtain the target mutants. Targeted mutagenesis technologies, such as TALEN, ZFN and CRISPR-Cas9 approaches, are powerful tools to generate target gene mutations. Comparedwith TALENs and ZFNs, the CRISPR-Cas9 system efficiently producesmutations and is easy to use.Sequencing of several gene clones from the mutant roots revealed that the CRISPRCas9 system continued to modify the genes during hairy root development, which suggests that the mutation efficiency would be increased given enough time for the development of the transgenic plants. The high efficiency of the target gene mutation can improve the research on gene function in soybean.Biallelic mutants can be detected easily using the PCR-RE assay. Biallelic mutants are the ideal materials for researching gene function. Compared to the inefficient and time-consuming transformation mediated by Agrobacterium tumefaciens (A. tumefaciens), transformation mediated by A. rhizogenes is easy, quick and efficient in soybean.Off-target activity is common using the CRISPR-Cas9 system. DecreasingsgRNA-Cas9 concentrations can increase on-target specificity in vitro? ? using double-nicking mediated by a Cas9 nickase mutant a shorter sgRNA seed (typically 17 or 18 nucleotides) complementary to thetargetthe best strategy is identification of gene-specific sgRNA seeds.T-DNA-induced mutagenesis has been widely applied in model plants such as Arabidopsis and rice. Successful T-DNA insertion mainly depends on efficient of A. tumefaciens-mediated transformation. In soybean, the creation of large numbers of mutants using T-DNA insertion is not feasible, as transformation efficiency mediated by A. tumefaciens is low in this speciesA total of 13,103,481 sgRNA seeds were predicted in soybean genes, of which5,631,730 were specific and 7,469,546 were non-specific. The huge quantity of nonspecific sgRNA seeds allows the targeting of two or more genes in one transformation in soybean (Fig. 6a). Off-target activity produces numerous mutations covering different genes in T0 transgenic soybeans. The resulting mutants can be segregated to produce unique mutations in the progeny, which, similar to the application of Ac/Ds transposons or Tnt1 retrotransposons in T-DNA transformations, improves mutation efficiency. In our study, the seeds of Glyma06g14180 or Glyma12g37050 were detected to produce two gene mutations (Glyma06g14180 and Glyma04g40610,Glyma12g37050 and Glyma09g00490)respectively in one transgenic plant. By exploiting off-target activity, the number oftransgenic soybean plants required to produce a saturated mutation library can be reduced dramatically (Fig. 6b).Figure 6. Non-specific synthetic guide RNA (sgRNA) seeds in soybean. (6a) Distribution of non-specific sgRNA seeds and the number of their target genes. More than 1 million sgRNA seeds were associated w approximately 100,000 sgRNA seeds were able to target three genes. sgRNA seeds having more than 100 target genes are not shown. (6b) Maximal gene coverage of non-specific sgRNA seeds. The non-specific sgRNA seeds were sorted by their target gene numbers before calculating the maximal gene coverageThankYou
更多相关文档CRISPRscan
CRISPRscan is a novel algorithm to predict gRNA efficiency.
Based on a large scale analysis of sgRNA mutagenesis activity in zebrafish, we established rules to predict sgRNA activity in vivo and build the CRISPRscan model integrating these rules. We independently validated with success our predictions using sgRNAs different from the large scale analysis.
Off-target predictions
CRISPRscan searches for potential genomic off-targets with the following rules.
AllAccording to
Nature Biotechnology 2013, potential off-targets can have a maximum of 2 mismatches with the sgRNA.
SeedWith the method published by
Science 2013, potential off-targets must match perfectly in their seed (12 nt 3' of the PAM sequence) and a maximum of 2 mismatches in the rest of the sgRNA. This rule is more stringent than the All method and therefore less off-targets are found.
CFD (Cutting Frequency Determination) Nature Biotechnology 2016 measured the cutting efficiency of potential off-targets and integrated them into the CFD score. Potential off-targets with up to 4 mismatches are scored with Doench et al. matrix.
AllPotential off-targets can have a maximum of 2 mismatches with the gRNA.
SeedThe experiments published by
Nature Methods 2017 support potential off-targets that must match perfectly in their seed (6 nt 3' of the PAM sequence) and a maximum of 2 mismatches in the rest of the gRNA. This rule is more stringent than the All method and therefore less off-targets are found.
gRNA generation
Detailed information can be found in our .
Oligo sequences
Oligo 1. sgRNA primerT7 promoterTarget sequenceTail annealing sequence
5’ TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA
alternative promoterSp6 promoter
5’ ATTTAGGTGACACTATAGANNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA
Oligo 2. Tail primerTailTail annealing sequence
5’ AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
Oligo sequences
Oligo 1. Tail primerT7 promoterTail sequence
5’ CCCTAATACGACTCACTATAGGTAATTTCTACTAAGTGTAGAT
Oligo 2. crRNA primerTarget sequence (reverse)Tail sequence
5’ NNNNNNNNNNNNNNNNNNNNNNNATCTACACTTAGTAGAAATTA
Oligo 1. Tail primerT7 promoterTail sequence
5’ CCCTAATACGACTCACTATAGGTAATTTCTACTCTTGTAGAT
Oligo 2. crRNA primerTarget sequence (reverse)Tail sequence
5’ NNNNNNNNNNNNNNNNNNNNNNNATCTACAAGAGTAGAAATTA
Oligo containing the target sequence are reported 5' to 3' ready to be ordered in the "Oligo" column, i.e. Cpf1 crRNA primer is reported reverse-complement of the target as shown above.CCC upstream of promoter are optional aiming to increase stability of oligo.
Gene annotation
Gene annotations were downloaded from
Plasmid: Cas9 with nanos 3’-UTR
Plasmid for targeting Cas9 expression into the germ line is available at .}