科普浅谈CRISPR/Cas12基因编辑技术Thefollowinghasevaluatedtonullormissing: ==>category[intemplate"site3/content/xiangqing

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CRISPR/Cas12系统于2018年被JenniferDoudna团队挖掘鉴定并在发表于Science杂志。CRISPR/Cas12系统由单一的RuvC结构域在远PAM端介导DNA切割，标签基因为Cas12，有的由单个crRNA介导，如CRISPR/Cas12a；有的由crRNA和tracrRNA共同介导(或融合后的sgRNA单独介导)，如CRISPR/Cas12b[1,2]。

CRISPR/Cas12系统的标签基因已有多达11种亚型(Cas12a-k)被鉴定，最近发现的CasX归类为Cas12e、CasY归类为Cas12d、Cas14归类为Cas12f、Casφ归类为Cas12j；其中研究最广泛的是Cas12a、Cas12b以及Cas12f[1]。

●Cas12a

●Cas12b

●Cas12f

Cas12f(又名Cas14a1)是依赖于crRNA和tracrRNA共同介导的(或融合后的sgRNA单独介导的)DNA核酸内切酶，能以PAM依赖的方式特异地剪切靶标dsDNA，使DNA双链断裂并生成黏性末端。与其他Cas12蛋白类似，Cas12f蛋白同样拥有针对ssDNA的反式剪切活性，不受PAM位点的限制，且双链或单链DNA靶标均能激活Cas12f的反式剪切活性。此外，相比于其他的Cas12蛋白，Cas12f蛋白的分子量普遍较小(400-700AA)。

◆CRISPR/Cas12基因编辑原理(以CRISPR/Cas12a为例）

●CRISPR/Cas12a的顺式剪切

●CRISPR/Cas12a的反式剪切

Cas12a/crRNA/DNA靶标三元复合物具有非特异性的ssDNA反式剪切活性。crRNA指导下，Cas12a识别PAM特异性识别和切割dsDNA，Cas12a发挥单链脱氧核糖核酸酶(ssDNase)活性，无差别裂解附近的ssDNA[5]。

CRISPR/Cas12a系统靶向识别剪切和反式剪切示意图[6]

◆基于CRISPR/Cas12核酸检测方法

2018年Doudna团队将恒温扩增技术与CRISPR/Cas12a相结合开发了一种检测系统，即DETECTR，该系统可用于对样本中的微量DNA进行快速、简便的即时检测[6]；同年，CellDiscovery杂志发布了HOLMES检测系统，该系统将PCR技术与CRISPR/Cas12相结合，不需要昂贵的试剂和特殊的仪器，效率高，成本低，易于进行核酸检测[7]。

DETECTR、HOLMES、Cas12aVDet检测[10]

◆CRISPR/Cas12结合高灵敏信号转导技术的核酸检测传感器

2020年Xu等人开发了一种基于CRISPR/Cas系统的增强型电化学DNA(E-DNA)传感器[11]。2021年Liu等人开发了一种基于CRISPR/Cas12a的电化学发光生物传感器，利用L-蛋氨酸稳定金纳米团簇作为高效电化学发光(ECL)元件实现电化学发光信号转换。这种生物传感器具有较高的选择性，在不需要核酸扩增的前提下实现了对人体血液样品中HPV-16的检测，整个检测可在70min内完成[12]。同年，Liang等人将CRISPR/Cas12系统表面增强拉曼光谱(SERS)相结合建立了SERS-CRISPR(S-CRISPR)检测平台，无需核酸扩增在30-40min完成SARS-CoV-2鼻咽子标本酸检测，是一种潜在的POCT检测方法[13]。2022年Yin等人设计了一种DNA/RNA嵌合杂交探针，并将其引入到CRISPR-Cas12a/SERS系统，该技术利用DNA/RNA嵌合发夹释放的ssRNA来分解纳米团簇，不需要核酸扩增，具有高选择性和高特异性[14]。

CRISPR/Cas12结合信号转导技术的核酸检测传感器[10]

◆蛋白检测

Dai等首次构建了基于CRISPR/Cas12a的蛋白检测平台，该平台将核酸适配体与靶标蛋白结合后，适配体作为Cas12a的识别底物启动其反式剪切活性，已成功用于转化生长因子β1的检测，且具有良好的灵敏度和特异性。除此之外，Zhao等开发了基于CRISPR/Cas12a的可用于检测蛋白质分析物的检测新平台，可用于临床样本中甲胎蛋白检测[6]。

CRISPR技术的快速发展为外泌体检测提供了新的思路和手段。Li等开发了基于CRISPR/Cas12a的高灵敏鼻咽癌CD109+外泌体检测平台，此外，Zhao等利用带有阻断器的适配体捕获CD63+外泌体，两者结合后适配体构象发生改变而释放阻断器，上清中的游离阻断器可激活Cas12a的反式剪切活性，进而释放荧光信号，从而实现靶标外泌体的检测[6]。Xing等使用抗体包被磁珠捕获外泌体，与适配体结合后经扩增产生可被Cas12a识别切割的长重复片段，然后非特异性切割体系中的荧光探针释放荧光信号，该平台可成功用于临床样本中外泌体表面蛋白nucleolin和程序性死亡配体1的定量检测[6]。

参考文献

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[2]韦涛,何滋林,纪庆超,祁艺新,常雯雯,隋宏书.CRISPR/Cas基因编辑系统研究进展[J].生命的化学,2019,39(01):39-45.

[3]王雪亮,胡晓波.CRISPR/Cas12系统在肿瘤检测中的应用[J].临床检验杂志,2023,41(03):225-228.

[4]董换哲,苑宁,张蕴哲etal.跨越式滚环等温扩增技术结合CRISPR/Cas12a定量检测海产品中的副溶血性弧菌[J].食品科学,1-10.

[5]孔康康.基于CRISPR-Cas12a技术的苹果茎沟病毒快速、低成本检测体系的开发与评价[D]:河南农业大学,2021.

[6]王雪亮,胡晓波.CRISPR/Cas12系统在肿瘤检测中的应用[J].临床检验杂志,2023,41(03):225-228.

[7]LiSY,ChengQX,WangJM,etal.CRISPR-Cas12a-assistednucleicaciddetection.CellDiscov,2018,4:20

[8]WangB,WangR,WangD,etal.Cas12aVDet:ACRISPR/Cas12a-basedplatformforrapidandvisualnucleicaciddetection.AnalChem,2019,91(19):12156-12161.

[9]DingX,YinK,LiZ,etal.SensitivequantitativedetectionofSARS-CoV-2inclinicalsamplesusingdigitalwarm-startCRISPRassay.BiosensBioelectron,2021,184:113218

[10]党生,张帅,翟景波.CRISPR/Cas12a系统：核酸检测的多功能工具[J].生物化学与生物物理进展:1-13.

[11]XuW,JinT,DaiY,etalSurpassingthedetectionlimitandaccuracyoftheelectrochemicalDNAsensorthroughtheapplicatior

ofCRISPRCassystems.BiosensBioelectron,2020,155:112100

[12]LiuPF,ZhaoKR,LiuZJ,etal.Cas12a-basedelectrochemiluminescencebiosensorfortargetamplification-freeDNAdetectionBiosensBioelectron,2021,176:112954

[13]LiangJ,TengP,XiaoW,etal.Applicationoftheamplification-freeSERS-basedCRISPR/Cas12aplatformintheidentificationofSARS-CoV-2fromclinicalsamples.JNanobiotechnology,2021,19(1):273

[14]YinB,ZhangQ,XiaX,etal.ACRISPR-Cas12aintegratedSERSnanoplatformwithchimericDNA/RNAhairpinguideforultrasensitivenucleicaciddetection.Theranostics,2022,12(13):5914-5930.

THE END

科普浅谈CRISPR/Cas12基因编辑技术Thefollowinghasevaluatedtonullormissing: ==>category[intemplate"site3/content/xiangqing

全球首款CRISPR基因编辑疗法获批，这种“基因剪刀“技术是如何诞生的？杜德纳dnarna微生物crispr

基因编辑技术研究利器—图形工作站/服务器硬件配置

gRNAdesignCRISPRScreensinglecellPerturbseq数据分析Life·Intelligence

CRISPR

CRISPR

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1.theuniverseofCRISPRCassequencesCRISPR-Cas序列的建模。 CRISPR相关蛋白语言模型 Cas9蛋白语言模型 指导RNA设计模型 基因编辑的产生 蛋白质生成受限。 用于表征的蛋白质的选择 sgRNA的设计 用于基础编辑的deaminases的设计 基因编辑器的特性 原文: Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences | bioRhttps://blog.csdn.net/weixin_46120256/article/details/138635902
2.研究人员建立蛋白工程化改造新方法和基于Cas12i的基因编辑新工具CRISPR-Cas基因组编辑技术在基因治疗、农作物经济性状改良及基础研究等领域均有多样化的应用，引领生物技术与应用的快速发展。自然界中广泛存在的天然CRISPR-Cas系统为新型基因编辑工具研发提供了丰富资源。然而，自然界微生物中发现的大多数Cas工具蛋白在哺乳动物细胞中的编辑效率较低，这限制了它们的应用，尤其是在生物https://baijiahao.baidu.com/s?id=1734341538486314607&wfr=spider&for=pc
3.CIDPCRISPRsgRNA设计模块CRISPR的重要性和应用前景,不需要再多罗嗦了。在CRISPR整个系统中,sgRNA是引导切割酶到达基因组指定位置的中间媒介,就是它负责识别目的基因进而发挥CRISPR切割等作用的。因而,sgRNA的设计是应用CRISPR系统的前提和关键。我整理了目前所能找到的26款sgRNA设计软件,其中有22款软件是需要用户选择背景数据集(即背景物种,通常https://www.jianshu.com/p/202f198fb188
4.CRISPRoffinder:aCRISPRguideRNAdesignandoffHowever, most of these tools can only design sgRNAs for the CRISPR/Cas system. In this study, a user-friendly standalone program named “CRISPR-offinder” was developed to provide researchers a tool for quick design of sgRNAs with minimal off-target effects for different CRISPR systems, https://www.ijbs.com/v13p1470.htm
5.CRISPRGuideDesignToolsandAlgorithmSTEMCELLTechnologiesThe CRISPR Guide Design Tool uses best practices and the latest computational tools to deliver the optimal CRISPR RNA (crRNA) or single guide RNA (sgRNA) sequence for every gene in the human and mouse genomes. Access the CRISPR Design Tools https://www.stemcell.com/crispr-guide-design-algorithm
6.CRISPRgRNADesigntoolCRISPR gRNA Design tool lets you design gRNA(s) to efficiently engineer your target and minimize off-target effects using ATUM Scoring Algorithms.https://www.atum.bio/eCommerce/cas9/input
7.CRISPRDesign custom gRNA CRISPR-Cas9 gRNA checker Species Input format Paste/Type input Upload file Enter up to 99 FASTA Sequences. Please enter sequences in standard FASTA formatting. This field is required. IDT RUO products are manufactured in accordance with ISO 9001 and are intended for research https://www.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE
8.pfcellsCRISPR Design Tool World’s Fastest & Easiest CRISPR Gene Knockout Design Tool Launch Design Tool Bioinformatics Tools CRISPR Design Tool OverviewBenefitsDataLaunch Overview The Best CRISPR Design Tool for Knockouts. Our powerful CRISPR software simplifies gRNA design. Choose from over 120,000 genomes http://www.synthego.com/products/bioinformatics/crispr-design-tool
9.engineeringusinganorthogonaltriwe followed the previously developed HI-CRISPR design20, where the homology donor sequences were integrated into the gRNA expression cassette. We found that the stable maintenance of the homology donor resulted in a further increase in CRISPRd efficiency: from 80% with Sg10 (Table1) to ~?98https://www.nature.com/articles/s41467-017-01695-x
10.CRISPRCRISPR-Cas9 sgRNA design and construction Single guided RNAs (sgRNA) targeting exons 29–30 of ITGA2 were designed using the web tool of UCSC Genome browser (https://genome.ucsc.edu/). The selected gRNAs were fulfilled the requirement as MIT guide specificity >60, high predicted cleavage https://bio-protocol.org/mv2/prep600
11.GitHubBase functions and classes for CRISPR gRNA design. Contribute to crisprVerse/crisprBase development by creating an account on GitHub.https://github.com/crisprVerse/crisprBase
12.EE-CRISP Design of CRISPR constructs Check out our new CRISPR Library Designer (CLD): batch design of sgRNA libraries Download the dockerized version now atCLD on Github 1. Select organism: [HELP] 2. Select target region by gene symbol or sequence: http://www.e-crisp.org/
13.CRISPRgRNA(guideRNA)DesignToolforEukaryoticPathogensbatch mode available here Gene tagging batch mode available here(?) Our CRISPR/Cas gRNA design tool has a new look!! Job Name: RNA guided nuclease selection:(?) SpCas9: 20nt gRNA, NGG PAM on 3' endSaCas9: 21nt gRNA, NNGRRT PAM on 3' endAsCpf1: 20nt gRNA, TTTN PAM on 5' endhttp://grna.ctegd.uga.edu/
14.DECKO:Singleoligo,dualDual excision CRISPR knockout design CRISPR can be used to delete genomic sequences, by cutting genomic DNA at two sites and relying on non-homologous end-joining (NHEJ) mechanism to repair the break (Fig. 1a). gRNAs are introduced to cells by a plasmid vector, either through transfection https://www.biomedcentral.com/1471-2164/16/846
15.Addgene:CRISPRReferencesandInformationIn collaboration with the labs who have depositedCRISPR plasmids, we've created a series of links and guides to help you use CRISPR in your lab. Learn More Guide to CRISPR technology Addgene Blog Posts How to Design Your gRNA for CRISPR Genome Editing: John Doench from the Broad Institute http://www.addgene.org/crispr/reference/
16.CRISPR/Cas9系统质粒载体列表262210 pnos-Fok1:dCas9-nos Fok1-dCas9 Insect Expression nanos Bullock CRISPRflydesign(unpublished) 62211 pAct-Fok1:dCas9 Fok1-dCas9 Insect Expression act5C Bullock CRISPRflydesign(unpublished) Purify A catalytically inactive Cas9 (dCas9) fused to an epitope tag(s) can be used to purify genhttp://www.biovector.net/product/133373.html
17.CRISPRdirect2016-12-14 CRISPRdirect shows restriction enzyme cutting sites - Sample 2016-09-09 Added 10 species - List 2016-08-30 Added Human Japanese Reference Genome JRGv1. 2016-06-14 Added 10 species - List 2015-10-05 CRISPRdirect supports 200+ species - List of species 2015-01-13 HTTPShttp://crispr.dbcls.jp/
18.OntargetandoffThe crisprDesign (GitHub link) package provides user-friendly functionalities to extract and score those sequences automatically via the addOnTargetScores function.4 On-targeting efficiency scores Predicting on-target cutting efficiency is an extensive area of research, and we try to provide in crisprhttp://bioconductor.org/packages/devel/bioc/vignettes/crisprScore/inst/doc/crisprScore.html
19.ECRISP:DesignofcustomgRNAconstructsBioinformatics methods can be used to find suitable target sites for the DSB in a systematic manner. We developed E-CRISP to design CRISPR constructs and provide the possibility to alter various design parameters systematically. A fast nucleotide indexing approach and the application of a binary intehttps://thenode.biologists.com/e-crisp-design-of-custom-grna-constructs/research/
20.SequencedeterminantsofimprovedCRISPRsgRNAdesigndifferent from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies. Footnoteshttp://doi.org/10.1101/gr.191452.115
21.CRISPR其脱靶评分公式与上述 2.1.5 Cas-OFFinder “CRISPR Design” 软件相同,但仅针对植物进行 该软件[30] 仅可评估 sgRNA 的脱靶效应(http:// sgRNA 设计。最大特色是搜寻sgRNA 靶标位点中是 否含限制性内切酶识别序列,以方便采用酶切法检 /cas-offinder/) ,由韩国首尔国立大 测基因组切割效率。 学(Seoul https://max.book118.com/html/2017/0920/134497738.shtm
22.CloudBenchling is a cloud-based platform for biotechnology research and development and the only biology-first platform for scientific data, collaboration, and insights.https://www.benchling.com/
23.CRISPRVisit the old version ofCRISPR-P Reference 1. Yang Lei, Li Lu, Hai-Yang Liu, Sen L, Feng Xing, Ling-Ling Chen*. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant, 2014, 7(9): 1494-1496.doi: 10.1093/mp/ssu044. http://crispr.hzau.edu.cn/
24.CRISPRCas9mediatedLAG3disruptioninCARWe obtained the first exon sequence ofLAG-3from NCBI and used the CRISPR Design Tool (http://crispr.mit.edu) to design sgRNAs. Oligonucleotides containing T7 promoter and 20 bp targeting sequences were synthesized as forward primer (Supplementary Table S1). T7-sgRNA PCR product was amplifiedhttps://journal.hep.com.cn/fmd/EN/10.1007/s11684-017-0543-6
25.CRISPRGPT:AnLLMAgentforAutomatedDesignofGeneAfter completing design tasks, CRISPR-GPT offers selected protocols based on the interaction history, including CRISPR system selection and delivery methods (See example in Figure 5 General Task 5). Finally, for the validation task, CRISPR-GPT utilizes external APIs, like Primer3, to assist usershttp://arxiv.org/html/2404.18021v1
26.CRISPRGuideRNADesign:MethodsandProtocolsSpringerLinkThis detailed volume focuses on the CRISPR-associated guide RNA and how it can be designed, modified, and validated for a broad repertoire of purposes. Beginning with a section on computational design of target-specific guide RNAs, the book continues by covering chemical modifications to alter guidhttps://www.springer.com/us/book/9781071606865
27.CRISPRHere we describe a web tool called CRISPR-ERA for automated genome wide sgRNA design. CRISPR-ERA can provide different sgRNA searching approaches for genome editing, such as Cas9 nuclease. In addition, CRISPR-ERA also generate sgRNAs for gene activation or repression using our large-scale databashttp://crispr-era.stanford.edu/
28.SureDesign,用于NGSCGHCRISPRFISH的定制设计安捷伦Agilent SureDesign 为 NGS、CGH、CRISPR 和 FISH 创建定制设计。对于 NGS,SureDesign 支持定制 SureSelect 和 HaloPlex 靶向序列捕获文库设计。对于 CGH,SureDesign 支持用于 CGH、ChIP-on-chip 和 DNA 甲基化的定制微阵列芯片设计。https://www.agilent.com/zh-cn/product/next-generation-sequencing/ngs-data-analysis-interpretation/suredesign-4301564
29.CRISPR/Cas9植物基因敲除试剂盒CRISPR Design:http://cbi.hzau.edu.cn/cgi-bin/CRISPR (2)选取 atttttcagGCTCTACCTAACCAGCAAACCGTAGATTATCCCAGCTTCAAGCTTGTCATTGTTGGTGATGGAGGCACAGgtacggt (3)输入序列信息(以拟南AtRAN1基因为例) (4)点submit,出现右侧结果; (5)根据左边不同Guide序列score的高低选取合适的Guide序列,score的高低并不代表敲https://www.biomart.cn/infosupply/31603201.htm
30.FrontiersCRISPRGeneTherapy:Applications,Limitations(31,57), CRISPR-design, CasOFFinder, and others (31). However, many of these tools are designed based on computational algorithms with varying parameters or rely on phenotypic screens that may be specific to cell types and genomes, generating appreciable noise and lack of generalizability acrosshttps://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2020.01387/full
31.anaccuratecelllineageusingCRISPRrecorders?eLifeComputer simulations reveal the potential and limitations of recently proposed CRISPR-based cell lineage recorders, and suggest how the recorders' design can be optimised to yield more accurate cell lineage trees.http://elifesciences.org/articles/40292