CN115851706A - 一种碱基编辑系统及其应用 - Google Patents

一种碱基编辑系统及其应用 Download PDF

Info

Publication number
CN115851706A
CN115851706A CN202111123978.3A CN202111123978A CN115851706A CN 115851706 A CN115851706 A CN 115851706A CN 202111123978 A CN202111123978 A CN 202111123978A CN 115851706 A CN115851706 A CN 115851706A
Authority
CN
China
Prior art keywords
sgrna
dna
artificial sequence
agg
tgg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111123978.3A
Other languages
English (en)
Inventor
吴宇轩
徐赛娟
程艳
赵飞燕
席在喜
刘明耀
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China Normal University
Bioray Laboratories Inc
Original Assignee
East China Normal University
Bioray Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by East China Normal University, Bioray Laboratories Inc filed Critical East China Normal University
Priority to CN202111123978.3A priority Critical patent/CN115851706A/zh
Priority to PCT/CN2022/120949 priority patent/WO2023046086A1/zh
Publication of CN115851706A publication Critical patent/CN115851706A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种碱基编辑系统及其应用。一种碱基编辑系统，其包含ABE8e碱基编辑器和如本发明所述的sgRNA文库；较佳地，所述ABE8e碱基编辑器的序列如SEQ ID NO:871所示。本发明的碱基编辑器在人细胞中靶向HBG启动子从而上调胎儿血红蛋白即γ‑珠蛋白的表达来治疗β血红蛋白病(如β地中海贫血、镰刀型细胞贫血症等疾病)。本发明提供的碱基编辑器在β血红蛋白病基因治疗领域具有重要的临床应用前景。

Description

一种碱基编辑系统及其应用
技术领域
本发明属于基因治疗领域，具体涉及一种sgRNA文库、含其的碱基编辑系统及其应用，还涉及一种通过高特异性ABE8e碱基编辑系统在β血红蛋白病治疗中的应用。
背景技术
地中海贫血(地贫)是全球分布最广、累及人群最多的一种单基因遗传病，每年有超过1,300,000名患有严重血红蛋白病症的患者出生。正常人血红蛋白(hemoglobin，Hb)中的珠蛋白有4种肽链，即α，β，γ和δ，根据珠蛋白肽链组合的不同而形成3种血红蛋白，即HbA(α2β2)，HbA2(α2δ2)和HbF(α2γ2)。HbA是成人红细胞中的主要血红蛋白，约占Hb中的95％，HbA2约占成人Hb中的2％，HbF是胎儿期及早期新生儿期的主要血红蛋白，在成人中，HbF占总血红蛋白的不到2％。人类血红蛋白的表达在发育过程中经历了两个转变：胚胎到胎儿和胎儿到成人，在出生后主要形式从胎肝中的HbF(α2γ2)转变为骨髓中的HbA(α2β2)。γ-globin是一种胎儿时期表达的珠蛋白，该珠蛋白具有和β-globin相似的功能，编码该蛋白的基因HBG(HBG1、HBG2)在贫血患者中序列完好，但到成年期表达便会被沉默。异体造血干细胞移植是目前根治“地贫”的方法，但花费巨大，且缺乏匹配的捐献者，许多患者无法使用造血干细胞移植，因此基因治疗正在成为新的治疗方式。
β-地中海贫血(β-Thalassemia，β-地贫)是一种由于β-珠蛋白(β-globin)亚基突变导致患者自身的成人血红蛋白(HbA)异常的遗传性疾病，是最常见的地贫类型之一。研究报道改善β-地贫临床症状的修饰作用可以通过重新激活HbF表达，β-地贫患者表型多样性是由于HbF含量具有明显的个体差异性，一般以0％～98％不等，HbF含量越高，患者贫血症状越轻。通过基因编辑造血干细胞促进了β地中海贫血和镰状细胞贫血病(SCD)治疗的创新策略，包括利用同源性定向DNA修复纠正SCD相关突变，通过强制启动子-LCR环或NHEJ介导的BCL11A编码HBG1和HBG2转录的阻遏因子，然而，这些方法还没有在患者中进行测试。
先前的研究发现，利用CRISPR/Cas9编辑技术可以重新激活胎儿血红蛋白HbF，提升γ珠蛋白的表达，代替有缺陷的β珠蛋白，是镰状细胞病(SCD)和β-地中海贫血的一种有希望的治疗策略。CRISPR/Cas9系统介导的基因编辑的本质是产生双链断裂，宿主细胞利用自身的非同源末端连接(nonhomologous end-joining，NHEJ)或基于同源重组(homologousend recombination repair，HDR)进行修复，但通常会在靶点处引入不可控的插入或缺失突变(Indels)，且存在非靶向切割产生较高的脱靶性，安全性较低。人类(Homo sapiens)大多数遗传疾病是由于碱基突变造成的，通过HDR对点突变的修复效率很低，而且在不分裂的细胞中基本上不发生HDR。碱基编辑技术(base editing)是在CRISPR/Cas9系统基础上发展起来的新型靶基因修饰技术，碱基编辑在基因组上直接对单个核苷酸进行突变，几乎不产生双链断裂(DSB)，也无需模板，是一种精确的基因编辑技术。
β-地中海贫血有超过300多种是由于突变造成的，目前基因编辑修正β-珠蛋白基因的方式，主要存在两个问题：一是以同源重组原位修复的方式引入正常表达的β-珠蛋白基因，通常因效率很低而受到限制；二是通过基因编辑的方式，删除突变位点或者碱基编辑修复某个位点，可能只针对一种或者几种类型的β-地中海贫血，普适性很低。
因此基于此需求，为了开发出能够同时治疗不同基因突变类型的β-地中海贫血，科学家们进一步开发了碱基编辑技术。2017年，美国华裔科学家David R.Liu课题组开发了高效诱导点突变的腺嘌呤单碱基编辑器(ABE)，腺嘌呤碱基编辑器(adenine base editor,ABE)能够使A·T转换为G·C，可以在不引入双链断裂的情况下实现高效的单碱基置换。碱基编辑技术，是通过融合脱氨酶产生的，sgRNA主要引导碱基编辑器到靶点位置，Cas9缺口酶(Cas9 nickase，nCas9)结合DNA双链并形成单链缺口，脱氨酶使非缺口链靶点附近腺嘌呤或者胞嘧啶脱氨，然后通过DNA复制引入碱基，达到碱基替换的目的。碱基编辑的方式很少造成indel，不需要依赖同源重组方式，具有安全高效的显著优势。在人类已知的致病单碱基突变中，有48％(约15000个)的突变是G突变成A，有14％(约4500个)的致病突变是A突变成G。单碱基编辑在基因敲除、敲入、碱基修复等领域得到了广泛的应用，其不断的发展在一定程度上纠正了一定比例的致病性SNP(single nucleotide polymorphism,SNP)。
单碱基编辑器自出现以来，因其安全高效、不依赖DNA双链断裂产生、无需供体DNA参与等优势不断发展优化，已被用来治疗地中海贫血、血友病等遗传疾病，包括在小鼠内耳的基因编辑，将是开展耳聋基因治疗的有效工具。随着单碱基编辑工具的研发，未来可能通过碱基的任意转化实现基因治疗。
2020年3月16日，David Liu团队发现ABE8e能够有效地产生天然突变，并通过编辑人细胞中BCL11A增强子或HBG启动子来上调胎儿血红蛋白的表达，这增强了腺嘌呤碱基编辑的有效性和适用性。目前还未有报道通过ABE8e碱基编辑器针对HBG非编码序列和编码序列进行高通量饱和筛选的策略。
发明内容
为了解决上述技术问题，本发明提供一种sgRNA文库、含其的碱基编辑系统以及一种非治疗目的的碱基编辑方法、提高γ珠蛋白RNA表达的方法、提高HbF表达的方法；以及所述碱基编辑系统的应用。本发明的碱基编辑器在人细胞中靶向HBG启动子从而上调胎儿血红蛋白即γ-珠蛋白的表达来治疗β血红蛋白病(如β地中海贫血、镰刀型细胞贫血症等疾病)，本发明提供的碱基编辑器在β血红蛋白病基因治疗领域具有重要的临床应用前景。
提升成人红细胞中的胎儿血红蛋白(HbF)水平为患有β-地中海贫血的患者提供临床益处，目前仍然是治疗镰状细胞病和地中海贫血患者的一个关键目标。为了在红细胞中鉴定到潜在可有效提升HbF水平的靶点，实验中使用最新优化的脱氧腺苷脱氨酶TadA-8e，通过ABE8e系统针对HBG启动子处的靶点进行高通量筛选。
为了有效识别可能在HBG1和HBG2启动子处诱导胎儿血红蛋白(HbF)的靶点，本发明使用了稳定表达的成人型(低HbF)HUDEP-2细胞，在人类γ-珠蛋白基因启动子区域设计了870个sgRNA进行了ABE8e的点突变饱和筛选。HbF染色后对高表达HbF的10％细胞和低表达HbF的10％细胞进行了流式分选，并通过深度测序分析了每个sgRNA的表达(即每个sgRNA的得分情况，其中得分较高的sgRNA见表1)。通过结果得出，对照sgRNAs在HbF高和低群体之间均匀分布。
根据深度测序结果对sgRNA进行排名，本发明从饱和筛选文库中挑选了排名比较靠前的sgRNA，这些sgRNA在高表达HbF细胞中富集，然后将单个sgRNA导入细胞中进行验证。首先在HUDEP-2细胞中进行验证，以1620为阳性对照，AAVS1为阴性对照，结果显示，大部分经过sgRNAs编辑的细胞γ-珠蛋白基因水平显著增加，证实了筛选的结果。
此外，进一步通过RNP电转的方式在CD34细胞中验证，sgRNA编辑的细胞显示出相似的γ-珠蛋白再激活水平。本发明的研究结果显示通过ABE8e高通量筛选HBG启动子可筛选出治疗血红蛋白新疗法的潜在靶标。ABE8e相比于其它编辑器脱靶率低，编辑效率高。且重要的是，针对HBG启动子区域进行筛选不会对细胞的分化和成熟产生有害的影响，其为最佳的位置。因此本专利在HBG(HBG1、HBG2)启动子上设计针对PAM位点(NGG)的饱和文库，用ABE8e编辑工具进行新位点的筛选来提升HbF表达水平。
本发明技术方案的一个方面提供一种靶向HBG启动子的sgRNA文库，其包含如SEQID NO:1、2和3所示的核苷酸序列中的一种或多种。
当所述sgRNA文库包含如SEQ ID NO:1、2和3所示的核苷酸序列中的一种时，所述sgRNA文库实质为一种sgRNA，本发明所述的sgRNA文库即为核苷酸序列为SEQ ID NO:1、2或3的sgRNA。
sgRNA405的序列如SEQ ID NO:1所示；sgRNA408的序列如SEQ ID NO:2所示；sgRNA411的序列如SEQ ID NO:3所示。
本发明中所述HBG为编码γ-globin蛋白的基因HBG1和HBG2。
本发明的sgRNA文库中，所述sgRNA优选位于病毒载体上，更优选所述病毒载体为慢病毒载体。
当sgRNA位于病毒载体时，本发明旨在保护含有SEQ ID NO:1-SEQ ID NO:870核苷酸序列的一种或多种的重组病毒载体例如重组慢病毒载体。
本发明的sgRNA文库较佳地还包含如SEQ ID NO:4-SEQ ID NO:59所示的核苷酸序列中的一种或多种。
本发明的sgRNA文库更佳地进一步包含如SEQ ID NO:59-SEQ ID NO:870所示的核苷酸序列中的一种或多种。
本发明技术方案的另一方面提供一种碱基编辑系统，其包含ABE8e碱基编辑器和如本发明所述的sgRNA文库。
本发明所述的ABE8e碱基编辑器中包含脱氧腺苷脱氨酶TadA-8e。
较佳地，所述ABE8e碱基编辑器的序列如SEQ ID NO:871所示。
本发明技术方案的另一方面提供一种药物组合物，所述药物组合物包括如本发明所述的碱基编辑系统。
较佳地，所述药物组合物还包括药物递送系统，比如所述药物递送系统为脂质体，外泌体、噬菌体或细菌微细胞。
本发明技术方案的另一方面提供一种基因工程细胞，所述基因工程细胞包含本发明所述的碱基编辑系统。
本发明所述的基因工程细胞的出发细胞较佳地为造血干细胞、造血祖细胞或红系祖细胞；和/或，所述出发细胞的来源为哺乳动物。
较佳地，所述出发细胞为CD34+造血干细胞或CD34+造血祖细胞；和/或，所述出发细胞的来源为人或鼠；例如人HUDEP-2细胞。
本发明技术方案的另一方面提供一种非治疗目的的碱基编辑方法，其通过本发明所述的碱基编辑系统、所述的药物组合物或所述的基因工程细胞对HBG启动子进行碱基编辑。
本发明的碱基编辑方法可为本领域常规，比如使用慢病毒感染的方式或使用RNP电转的方式，其中，在使用RNP电转时可通过化学修饰sgRNA，比如在sgRNA的3’端和5’端各加上3个硫代和甲氧基进行修饰。
本发明技术方案的另一方面提供一种非治疗目的的提高γ珠蛋白RNA表达的方法，其包括如本发明所述的碱基编辑方法。
本发明技术方案的另一方面提供一种非治疗目的的提高HbF表达的方法，其包括本发明所述的碱基编辑方法。
本发明中，所述HbF指的是HbF(α2γ2)血红蛋白。
本发明技术方案的另一方面提供一种如本发明所述的sgRNA文库、如本发明所述的碱基编辑系统、如本发明所述的药物组合物或者如如本发明所述的基因工程细胞在制备碱基编辑的药物中的应用。
较佳地，所述碱基编辑的药物为治疗遗传性疾病的药物。
更佳地，所述遗传性疾病为地中海贫血。
在某一较佳实施方案中，所述遗传性疾病为β地中海贫血。
本发明技术方案的另一方面提供一种如本发明所述的sgRNA文库、如本发明所述的碱基编辑系统、如本发明所述的药物组合物或者如本发明所述的基因工程细胞在制备碱基编辑工具或碱基编辑模型中的应用。
本发明所用试剂和原料均市售可得。
本发明所述的药物递送系统为狭义的递送系统，比如脂质体。
本发明的积极进步效果在于：
通过最新的高效ABE8e碱基编辑系统以及最新优化的脱氧腺苷脱氨酶TadA-8e高通量筛选HBG1和HBG2启动子区域新靶点的饱和文库，所述文库包括870个特异靶向HBG基因启动子区域的高特异性sgRNA。本发明的碱基编辑器在人细胞中靶向HBG启动子从而上调胎儿血红蛋白即γ-珠蛋白的表达来治疗β血红蛋白病(如β地中海贫血、镰刀型细胞贫血症等疾病)。本发明提供的碱基编辑器在β血红蛋白病基因治疗领域具有重要的临床应用前景。
附图说明
图1为LentiABE8e-Blast慢病毒载体的示意图。
图2为通过Sanger测序显示已构建成功稳定表达ABE8e的HUDEP-2细胞系，相对于未编辑细胞，编辑后的细胞可高达70％以上的基因组编辑效率。
图3为通过流式分析检测ABE8e系统对HUDEP-2细胞红系分化能力的影响。
图4为通过RT-qPCR显示HUDEP-2细胞感染慢病毒红系分化后，相对于未编辑细胞，编辑后的细胞内γ-珠蛋白mRNA相对于β珠蛋白mRNA的水平大部分显著提高(数字代表不同sgRNA的代表序号)。
图5显示了通过RT-qPCR显示RNP递送ABE8e/sgRNA在CD34+HSPCs进行高效的编辑，红系分化后γ-珠蛋白相对β-珠蛋白表达水平升高。
图6显示了通过流式分析检测CD34+造血干细胞红系分化后去核率水平。
图7显示了通过RT-qPCR显示β地贫病人CD34+造血干细胞RNP递送ABE8e/sgRNA后γ-珠蛋白相对β-珠蛋白表达水平升高。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1
1、构建sgRNA筛选文库。
针对γ-珠蛋白基因组上的HBG1和HBG2启动子处设计sgRNA，同时设计在基因组上无靶点的对照sgRNA，并包含之前已知的位点可以显著提升γ-珠蛋白表达的sgRNA(由金斯瑞公司合成)。将合成的sgRNA文库克隆到BsmBI酶切后的phko9慢病毒载体(addgeng官网)中，通过病毒包装获得病毒颗粒，用于感染HUDEP-2细胞。载体构建完成后，将载体文库使用感受态，扩增培养并收集菌落，并进行扩大培养，最后抽提质粒，即得到了扩增后的载体文库。
sgRNA文库慢病毒包装：扩增后的gRNA文库在慢病毒包装载体的辅助下转染293T细胞(上海邦耀生物有限公司)，进行慢病毒的包装与浓缩。
2、构建ABE8e稳定表达的HUDEP-2细胞系。
HUDEP-2细胞是永生性的人造血干祖细胞系，在体外经诱导可发生红系分化，获得HbA高表达和HbF低表达的红细胞，是研究血红蛋白转换的理想细胞模型。
构建lentiABE8e-Blast慢病毒表达载体(其图谱如图1所示)，用表达ABE8e的慢病毒感染HUDEP-2细胞(参考Kurita R,Suda N,Sudo K,Miharada K,Hiroyama T,et al.(2013)Establishment of Immortalized Human Erythroid Progenitor Cell LinesAble to Produce Enucleated Red Blood Cells.PLoS ONE8(3):e59890.doi:10.1371/journal.pone.0059890)，用药物blast筛选5天，获得稳定表达化脓性链球菌ABE8e(TadA-8e)的HUDEP-2细胞系。
在进行筛选之前，首先验证ABE8e蛋白(其序列如SEQ ID NO:871所示)的靶向活性，通过使用来自David Liu文章(参考Phage-assisted evolution of an adenine baseeditor with improved Cas domain compatibility and activity)中的阳性对照sgRNA，比如-198(即为sgRNA文库中的sgRNA418)，-175(即为sgRNA文库中的sgRNA424)，Site1在ABE8e-HUDEP-2细胞系中诱变靶位点，并且也选用了文库中不同位置的sgRNA，将选择的sgRNA构建到phko9慢病毒载体进行验证。包装病毒后感染ABE8e-HUDEP-2细胞系，在感染细胞后5天就可产生高达70％以上的编辑效率(如图2所示)，此外本申请还检测了该系统对其细胞红系分化能力的影响，结果表明，细胞中引入ABE8e蛋白未影响细胞的红系分化能力(如图3所示)。本申请包括两种方式将sgRNA和ABE8e导入细胞中，一种是通过慢病毒感染的方式，一种是通过RNP电转的方式。经过两种方式编辑的细胞，一般是在5天后提取基因组，设计距离编辑位点200bp左右远的引物，来检测编辑效率。
3、sgRNA文库在HUDEP-2细胞中筛选。
将HBG的sgRNA文库克隆到phko9慢病毒载体中，生成了一个编码870个gRNA和一个嘌呤霉素抗性的慢病毒载体文库，以0.3的感染复数感染HUDEP-2-ABE8e细胞，并在嘌呤霉素中筛选感染细胞7天，然后诱导红细胞分化。在红细胞成熟培养后，此时参与血红蛋白转换的位点被编辑后会影响细胞中HbF的表达量。用HbF抗体染色通过FACS分选HbF表达量最高和最低的各10％的细胞，对筛选中富集表达的细胞进行慢病毒载体的PCR扩增，该扩增片段包含载体所携带的gRNA序列，通过深度测序对这些细胞中的870个sgRNA进行分析，其中有59个得分较高(参考表1)，可以得到富集细胞中sgRNA的富集度，从而寻找到相应的sgRNA序列，这些位点很有可能参与HbF提升过程，因此可作为深入研究的靶点。由于sgRNA文库是由多个不同的sgRNA载体混合而成，因此本申请获得的慢病毒是混合型病毒库，每个病毒携带单个gRNA载体。对筛选的目的细胞进行病毒感染预实验，得到细胞在较低的MOI(30％)时感染百分数(确保每个细胞最多进入一个慢病毒)，以确认感染时sgRNA文库的病毒用量(检测病毒滴度在30％的情况下，细胞量为3万，感染病毒量为(10μL)。
如表1所示，根据深度测序的结果，分析高表达10％HbF与低表达10％HbF组相比显着富集的sgRNA在细胞中的富集情况。在使用MAGeCK的稳健排序整合(robust rankaggregation，RRA)方法和默认参数生信分析时，设置一定的阈值，FDR<0.05和log2Fold-Change>log2(1.5)的过滤器用于定义上调的候选sgRNA。然后对sgRNA进行排名，此排名代表sgRNA的相对富集情况，即排名超过一定阈值，就有激活HbF表达的效果。
表1：得分较高的sgRNA
sgRNA编号 得分 SEQ ID NO: sgRNA编号 得分 SEQ ID NO:
sgRNA 541 34.395 4 sgRNA 142 3.9528 32
sgRNA 1066 23.539 5 sgRNA 418(对照) 3.5567 33
sgRNA 689 21.34 6 sgRNA 65 3.3742 34
sgRNA 562 21.187 7 sgRNA 225 3.232 35
sgRNA 32 15.82 8 sgRNA 351 3.1693 36
sgRNA 62 14.906 9 sgRNA 31 2.7039 37
sgRNA 944 12.671 10 sgRNA 342 2.5178 38
sgRNA 445 12.449 11 sgRNA 661 2.4697 39
sgRNA 234 11.072 12 sgRNA 974 2.4628 40
sgRNA 543 10.548 13 sgRNA 470 2.373 41
sgRNA 616 9.6405 14 sgRNA 931 2.3715 42
sgRNA 621 9.0611 15 sgRNA 309 2.271 43
sgRNA 1058 8.4345 16 sgRNA 479 2.0708 44
sgRNA 90 8.4343 17 sgRNA 1021 2.0362 45
sgRNA 409 8.0433 18 sgRNA 986 2.0039 46
sgRNA 118 7.882 19 sgRNA 312 1.5273 47
sgRNA 347 7.6729 20 sgRNA 14 1.495 48
sgRNA 242 7.6281 21 sgRNA 513 1.2857 49
sgRNA 1061 7.2964 22 sgRNA 706 1.1925 50
sgRNA 658 7.2245 23 sgRNA 405 1.1237 1
sgRNA 1065 7.2136 24 sgRNA 223 1.0884 51
sgRNA 955 6.8254 25 sgRNA 496 1.039 52
sgRNA 626 6.7586 26 sgRNA 245 1.0353 53
sgRNA 653 6.5252 27 sgRNA 424(对照) 0.7537 54
sgRNA 27 6.4956 28 sgRNA 120 0.70886 55
sgRNA 520 6.45 29 sgRNA 657 0.68753 56
sgRNA 665 6.3462 30 sgRNA 370 0.68623 57
sgRNA 987 5.9911 31 sgRNA 558 0.57447 58
sgRNA 408 4.303 2 sgRNA 491 4.004 59
实施例2候选sgRNA的验证
1、在HUDEP2-ABE8e细胞中对候选sgRNA进行验证。
将排名靠前的sgRNA导入高表达ABE8e蛋白的HUDEP-2细胞中，以sgRNA1620(sgRNA1620参考Therapeutic base editing of human hematopoietic stem cells)(其序列如SEQ ID NO:872所示)的sgRNA转染组为阳性对照，以基因组上无靶点的AAVS1 sgRNA(如SEQ ID NO:873所示)转染组为阴性对照。将sgRNA1620作为阳性对照是因为文献中报道通过单碱基编辑工具编辑该位点，具有很高的编辑效率，并显著提升γ-珠蛋白水平。本申请将选择的sgRNA构建到phko9慢病毒载体上，并通过293T包装产生病毒，以相同病毒量(20万细胞培养在24孔板中，感染10μL病毒量)感染HUDEP-2细胞，细胞经红系诱导分化后，用RT-qPCR检测γ珠蛋白RNA水平表达量的变化。
RT-PCR的结果显示，编辑后的细胞内γ-珠蛋白的mRNA相对于未编辑的细胞的mRNA平均表达水平显著提升。在红系分化后，通过FACS来检测细胞内HbF的表达量，编辑后的细胞HbF的平均表达量相比于未编辑的细胞HbF的平均表达量显著提高(如图4所示)。基于此结果说明通过慢病毒感染对正常人的HUDEP-2细胞进行编辑可以重新激活细胞内γ-珠蛋白基因的表达，使编辑后的红系分化后HUDEP-2细胞内的γ-珠蛋白和HbF表达水平提高。
2、在CD34+细胞中对候选sgRNA进行下一步验证。
与慢病毒载体相比，电穿孔(ribonucleoprotein，RNP)是比较常用的物理递送方法，对ABE8e蛋白和sgRNA直接进行递送，在人的CD34+HSPCs细胞中具有更高的转导效率，可以最大程度地降低脱靶效应，而且RNP可以在细胞内快速降解，提高了安全性。实验利用电穿孔的方式将ABE8e蛋白和经化学修饰(3’端和5’端各加上3个硫代和甲氧基)的sgRNA通过RNP的形式递送到CD34+HSPCs(上海邦耀生物有限公司)中，靶向γ-珠蛋白基因启动子区以治疗β-地中海贫血。实验中以1620为阳性对照，AAVS1 sgRNA为阴性对照，在红系分化后RT-qPCR检测γ珠蛋白基因的表达，在第18天用Hochest染色后流式分析检测细胞的去核率。
本申请发现，通过合成针对γ-珠蛋白基因启动子区位点的sgRNAs，并经过化学修饰其两端，利用RNP方式将ABE8e蛋白和sgRNA以核糖核蛋白复合物的形式递送到CD34+造血干/祖细胞中进行编辑，在无分选的情况下，CD34+细胞中的平均编辑效率可以达到80％以上。经过红系分化，编辑后的细胞内γ-珠蛋白mRNA相对于β-样珠蛋白mRNA的平均表达水平普遍升高(如图5所示)，尤其是sg RNA408、sg RNA405和sg RNA411位点，其γ-珠蛋白基因表达相对于β-珠蛋白平均含量显著提高(如图5所示)，脱核率也在40％左右(如图6所示)。
基于上述结果表明，利用核糖核蛋白的递送方式可以高效编辑CD34+造血干/祖细胞的同时避免了慢病毒载体所带来的安全隐患，为基因编辑治疗β-地中海贫血提供新的方法和实验基础。
3、选择β-地中海贫血的病人来源的CD34+细胞对调控靶点的sgRNA进行验证。
研究表明，升高HbF可一定程度上改善β-地中海贫血病人。因此为了进一步证明ABE8e/sgRNA编辑人的CD34+HSPCs可以诱导γ-珠蛋白的表达，我们选择将候选靶点的sgRNA电转β-地中海贫血病人来源的CD34+细胞中，然后进行体外分化，红系分化后检测γ珠蛋白的表达。结果表明，经过编辑的病人CD34+HSPCs细胞红系分化18天后，其γ-珠蛋白mRNA相对于β样-珠蛋白mRNA平均表达水平显著提升(如图7所示)。
这些结果表明ABE8e/sgRNA RNP对β-地中海贫血病人CD34+HSPCs进行基因编辑，提高了编辑后的细胞内的γ-珠蛋白的表达。因此，ABE8(RNP)介导的HBG1和HBG2启动子中结合位点的破坏是治疗β-地中海贫血的潜在可行和安全的治疗策略。
SEQUENCE LISTING
<110> 华东师范大学
上海邦耀生物科技有限公司
<120> 一种碱基编辑系统及其应用
<130> P21014714C
<160> 873
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA405
<400> 1
cttgtcaagg ctattggtca agg 23
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA408
<400> 2
aggcaaggct ggccaaccca tgg 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA411
<400> 3
gctaaactcc acccatgggt tgg 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA541
<400> 4
ctttatgatt tgatggagcc agg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1066
<400> 5
gctacttggg aggctgagga agg 23
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA689
<400> 6
cacaaagggt tttatgttga ggg 23
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA562
<400> 7
catctgcctt acttagtgca agg 23
<210> 8
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA32
<400> 8
gccgctggtt gttcctttta tgg 23
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA62
<400> 9
atagatgaga gtagtagagt agg 23
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA944
<400> 10
cactggagct acagacaaga agg 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA445
<400> 11
atgctttagc tttaaactac agg 23
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA234
<400> 12
ccccttcaag cactagtcac tgg 23
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA543
<400> 13
ttctccaaac gtcttctgcc tgg 23
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA616
<400> 14
agacctcatt gtaagcttct agg 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA621
<400> 15
atacccttaa gtctacagac tgg 23
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1058
<400> 16
tcacccaggc tggagtgcag tgg 23
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 17
ctcactgtgt cctcaccggg agg 23
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA409
<400> 18
ggcaaggctg gccaacccat ggg 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA118
<400> 19
ctgtgagatc aaaatgtagt ggg 23
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA347
<400> 20
aagcgacctg gacttttgcc agg 23
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA242
<400> 21
gtcttcagcc tacaacatac agg 23
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1061
<400> 22
ggagtgcagt ggtatgatct cgg 23
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA658
<400> 23
gctgtgctgc aatccaggga agg 23
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1065
<400> 24
gaaggagaat tgcttgaacc tgg 23
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA955
<400> 25
ataggtccag gatttttgat ggg 23
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA626
<400> 26
aaaagttcct tagtaaagta agg 23
<210> 27
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA653
<400> 27
tgcagcacag cagcgaggaa ggg 23
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA27
<400> 28
ctatatggcc taaaaagaga agg 23
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA520
<400> 29
ataacttcct gatgttgcca tgg 23
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA665
<400> 30
atggggaaag gttcagtgaa ggg 23
<210> 31
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA987
<400> 31
catggaaaac aactcttaag agg 23
<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA142
<400> 32
ttagcttttc acagcccttg tgg 23
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA418
<400> 33
atatttgcat tgagatagtg tgg 23
<210> 34
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA65
<400> 34
tattttatac tgaaacatag ggg 23
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA225
<400> 35
gctttgaggg aagttaacat agg 23
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA351
<400> 36
ggaaggaagg accctgtgcc tgg 23
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA31
<400> 37
accataaaag gaacaaccag cgg 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA342
<400> 38
actgttcttg tcaatctcac agg 23
<210> 39
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA661
<400> 39
gtgctgcaat ccagggaagg ggg 23
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA974
<400> 40
tatgtgctgg ctgatgaccc agg 23
<210> 41
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA470
<400> 41
gtgtgctggc tgatgaccca ggg 23
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA931
<400> 42
attaagcagc agtatcctct tgg 23
<210> 43
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA309
<400> 43
gcactgtgac aagctgcatg tgg 23
<210> 44
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA479
<400> 44
aaatgctagg ttcacttctc agg 23
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1021
<400> 45
ttatatctgc tcaaagtgtt agg 23
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA986
<400> 46
acataagaca tgtatttgca tgg 23
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA312
<400> 47
cagttcactc agctgggcaa agg 23
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA14
<400> 48
ttataggtaa aagtgtgcca tgg 23
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA513
<400> 49
ggatgtgcag gcttgttaca tgg 23
<210> 50
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA706
<400> 50
tccagtcacc atcttctgcc agg 23
<210> 51
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA223
<400> 51
gttaacatag gcttgattct ggg 23
<210> 52
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA496
<400> 52
tctggcatta taatctagca agg 23
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA245
<400> 53
tgaggaacaa ttgaaacatt tgg 23
<210> 54
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA424
<400> 54
gtggggaagg ggcccccaag agg 23
<210> 55
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA120
<400> 55
acattttgat ctcacagtgc tgg 23
<210> 56
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA657
<400> 56
cgctgctgtg ctgcaatcca ggg 23
<210> 57
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA370
<400> 57
ccacattcac cttgccccac agg 23
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA558
<400> 58
ttgtaaacct gagataaaca tgg 23
<210> 59
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA491
<400> 59
aacagggtag gaagtattta tgg 23
<210> 60
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 60
caaaagcaaa aattgaccaa tgg 23
<210> 61
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 61
ttagtttaat tagattccat tgg 23
<210> 62
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 62
ggtagtttct tttgctgagc agg 23
<210> 63
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 63
tagattgtct ttcgctctga tgg 23
<210> 64
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 64
gcgaaagaca atctacagaa tgg 23
<210> 65
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 65
cgaaagacaa tctacagaat ggg 23
<210> 66
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 66
cctttgtcag aagaatagat tgg 23
<210> 67
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 67
ccaatctatt cttctgacaa agg 23
<210> 68
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 68
tcttgtttac tctggatatt agg 23
<210> 69
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 69
tttaagtttc ttgtttactc tgg 23
<210> 70
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 70
aaaaaaaccc attaaaacca tgg 23
<210> 71
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 71
aagtgtgcca tggttttaat ggg 23
<210> 72
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 72
aaagtgtgcc atggttttaa tgg 23
<210> 73
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 73
gatatgcagg tttgttttat agg 23
<210> 74
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 74
ggtacacgta caggatatgc agg 23
<210> 75
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 75
aaattcaggg gtacacgtac agg 23
<210> 76
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 76
tacgtgtacc cctgaattta agg 23
<210> 77
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 77
tactgttacc ttaaattcag ggg 23
<210> 78
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 78
ttactgttac cttaaattca ggg 23
<210> 79
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 79
tttactgtta ccttaaattc agg 23
<210> 80
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 80
aaattaaaag aaaaatgcca tgg 23
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 81
gtaactttct gacattgcca tgg 23
<210> 82
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 82
tgtcagaaag ttaccctata tgg 23
<210> 83
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 83
ttctcttttt aggccatata ggg 23
<210> 84
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 84
cttctctttt taggccatat agg 23
<210> 85
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 85
tatttatgcc ttctcttttt agg 23
<210> 86
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 86
atgatatgtt aaacaagttt tgg 23
<210> 87
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 87
catcaagaaa taaccataaa agg 23
<210> 88
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 88
aaggaacaac cagcggccct cgg 23
<210> 89
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 89
aggaacaacc agcggccctc ggg 23
<210> 90
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 90
ggaacaacca gcggccctcg ggg 23
<210> 91
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 91
agagcagccc cgagggccgc tgg 23
<210> 92
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 92
cataggcaga gcagccccga ggg 23
<210> 93
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 93
gcataggcag agcagccccg agg 23
<210> 94
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 94
taaacgaatg actactgcat agg 23
<210> 95
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 95
agaaagtgca tattctgaaa cgg 23
<210> 96
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 96
ggtagtgact gcatttgaag tgg 23
<210> 97
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 97
gtagtgactg catttgaagt ggg 23
<210> 98
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 98
gtgggacctt tatgatatga tgg 23
<210> 99
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 99
ctggctccat catatcataa agg 23
<210> 100
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 100
ctttatgata tgatggagcc agg 23
<210> 101
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 101
atggagccag gcagagatgt tgg 23
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 102
acttctccaa catctctgcc tgg 23
<210> 103
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 103
ttggagaagt tcctgaaaga agg 23
<210> 104
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 104
agaagttcct gaaagaagga agg 23
<210> 105
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 105
gaagttcctg aaagaaggaa ggg 23
<210> 106
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 106
cacatgccct tccttctttc agg 23
<210> 107
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 107
agggcatgtg ccaaattctg agg 23
<210> 108
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 108
tgtgccaaat tctgaggctg agg 23
<210> 109
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 109
ttctcctcag cctcagaatt tgg 23
<210> 110
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 110
ctgtatgtaa agacattaca agg 23
<210> 111
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 111
aagcatgttg aagtaaaaat agg 23
<210> 112
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 112
agaaatcaaa gttagagaga agg 23
<210> 113
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 113
gaaatcaaag ttagagagaa ggg 23
<210> 114
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 114
aaagttagag agaagggcgc agg 23
<210> 115
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 115
aagggcgcag gcttattatt tgg 23
<210> 116
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 116
agggcgcagg cttattattt ggg 23
<210> 117
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 117
ggtattttat actgaaacat agg 23
<210> 118
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 118
gtattttata ctgaaacata ggg 23
<210> 119
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 119
atactgaaac ataggggacg agg 23
<210> 120
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 120
tactgaaaca taggggacga ggg 23
<210> 121
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 121
actgaaacat aggggacgag ggg 23
<210> 122
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 122
ttgcaaacct gagataaaca tgg 23
<210> 123
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 123
ctacagacca tgtttatctc agg 23
<210> 124
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 124
aaataagctt gggttttgag tgg 23
<210> 125
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 125
aaggagaaaa aaataagctt ggg 23
<210> 126
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 126
gaaggagaaa aaaataagct tgg 23
<210> 127
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 127
ctgagtaagg cagatgaaga agg 23
<210> 128
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 128
catctgcctt actcagtgca agg 23
<210> 129
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 129
atagcacctt gcactgagta agg 23
<210> 130
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 130
tataacaaaa tacaaaagac tgg 23
<210> 131
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 131
ataacaaaat acaaaagact ggg 23
<210> 132
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 132
acaaaataca aaagactggg tgg 23
<210> 133
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 133
gttttttctc acatttctga agg 23
<210> 134
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 134
catttctgaa ggctgactcg tgg 23
<210> 135
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 135
ctgaaggctg actcgtggtc agg 23
<210> 136
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 136
tgaaggctga ctcgtggtca ggg 23
<210> 137
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 137
cgtggtcagg gttctcttcc tgg 23
<210> 138
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 138
ttctcttcct ggattgcaga tgg 23
<210> 139
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 139
tcttcctgga ttgcagatgg cgg 23
<210> 140
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 140
aagtccgcca tctgcaatcc agg 23
<210> 141
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 141
acttctcact gtgtcctcac cgg 23
<210> 142
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 142
cttctcactg tgtcctcacc ggg 23
<210> 143
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 143
atttctcttt cctcccggtg agg 23
<210> 144
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 144
tcaccgggag gaaagagaaa tgg 23
<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 145
aagccatttc tctttcctcc cgg 23
<210> 146
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 146
ttgtctctct gcttctgata agg 23
<210> 147
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 147
taaggaaaat aattttatga tgg 23
<210> 148
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 148
aaggaaaata attttatgat ggg 23
<210> 149
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 149
aggaaaataa ttttatgatg ggg 23
<210> 150
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 150
gaggcttttg aaaagtaatt agg 23
<210> 151
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 151
aaaagcctcc cccacagata agg 23
<210> 152
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 152
actgacctta tctgtggggg agg 23
<210> 153
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 153
ccaactgacc ttatctgtgg ggg 23
<210> 154
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 154
cccccacaga taaggtcagt tgg 23
<210> 155
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 155
cccaactgac cttatctgtg ggg 23
<210> 156
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 156
ccccacagat aaggtcagtt ggg 23
<210> 157
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 157
tcccaactga ccttatctgt ggg 23
<210> 158
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 158
ctcccaactg accttatctg tgg 23
<210> 159
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 159
agataaggtc agttgggagt tgg 23
<210> 160
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 160
gataaggtca gttgggagtt ggg 23
<210> 161
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 161
ataaggtcag ttgggagttg ggg 23
<210> 162
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 162
gattcagcat gtgaattttg agg 23
<210> 163
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 163
tttgaggaga cacaaacatt agg 23
<210> 164
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 164
agtttaatat gatggtgtta cgg 23
<210> 165
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 165
taagtaagag tttaatatga tgg 23
<210> 166
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 166
taatggaatt tttgtatagt tgg 23
<210> 167
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 167
taattggaag gcttacttaa tgg 23
<210> 168
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 168
tttgtacaac attaattgga agg 23
<210> 169
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 169
agaatttgta caacattaat tgg 23
<210> 170
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 170
actgtgagat caaaatgtag tgg 23
<210> 171
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 171
tgtaagaaat gaatcagcag agg 23
<210> 172
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 172
tctgggggag gtggggactt agg 23
<210> 173
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 173
gtaagactct gggggaggtg ggg 23
<210> 174
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 174
tgtaagactc tgggggaggt ggg 23
<210> 175
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 175
ttgtaagact ctgggggagg tgg 23
<210> 176
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 176
tcattgtaag actctggggg agg 23
<210> 177
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 177
tcccccagag tcttacaatg agg 23
<210> 178
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 178
acctcattgt aagactctgg ggg 23
<210> 179
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 179
gacctcattg taagactctg ggg 23
<210> 180
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 180
agacctcatt gtaagactct ggg 23
<210> 181
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 181
gagacctcat tgtaagactc tgg 23
<210> 182
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 182
gattggtcac agcatttcaa ggg 23
<210> 183
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 183
agattggtca cagcatttca agg 23
<210> 184
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 184
gtgaccaatc tgcacacttg agg 23
<210> 185
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 185
tgaccaatct gcacacttga ggg 23
<210> 186
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 186
atgccctcaa gtgtgcagat tgg 23
<210> 187
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 187
aatctgcaca cttgagggca tgg 23
<210> 188
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 188
atctgcacac ttgagggcat ggg 23
<210> 189
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 189
gggattaatt tcactttctt agg 23
<210> 190
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 190
cactttctta ggcatccaca agg 23
<210> 191
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 191
actttcttag gcatccacaa ggg 23
<210> 192
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 192
aggaggaata ctatttctaa tgg 23
<210> 193
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 193
agcaattcta ttttattagg agg 23
<210> 194
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 194
tgtagcaatt ctattttatt agg 23
<210> 195
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 195
taggaattca aggtttagtc agg 23
<210> 196
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 196
aaccttgaat tcctaaagct tgg 23
<210> 197
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 197
ttccaagctt taggaattca agg 23
<210> 198
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 198
gggaaagtgt tccaagcttt agg 23
<210> 199
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 199
caaggatggt tcttaaggaa ggg 23
<210> 200
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 200
gcaaggatgg ttcttaagga agg 23
<210> 201
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 201
agtagcaagg atggttctta agg 23
<210> 202
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 202
ttgcagctga gtagcaagga tgg 23
<210> 203
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 203
ttgattgcag ctgagtagca agg 23
<210> 204
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 204
ctgcaatcaa tccagccccc agg 23
<210> 205
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 205
ttcagtgaag acctgggggc tgg 23
<210> 206
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 206
aaggttcagt gaagacctgg ggg 23
<210> 207
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 207
aaaggttcag tgaagacctg ggg 23
<210> 208
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 208
aaaaggttca gtgaagacct ggg 23
<210> 209
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 209
gaaaaggttc agtgaagacc tgg 23
<210> 210
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 210
gttttggaag agatgggaaa agg 23
<210> 211
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 211
acagatgttt tggaagagat ggg 23
<210> 212
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 212
aacagatgtt ttggaagaga tgg 23
<210> 213
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 213
cttctcagaa acagatgttt tgg 23
<210> 214
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 214
tgagaagtcc tgtcctatag agg 23
<210> 215
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 215
aagaaagacc tctataggac agg 23
<210> 216
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 216
gtgggaagaa agacctctat agg 23
<210> 217
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 217
atagaggtct ttcttcccac cgg 23
<210> 218
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 218
tggtgtagga gaaatccggt ggg 23
<210> 219
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 219
atggtgtagg agaaatccgg tgg 23
<210> 220
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 220
taaatggtgt aggagaaatc cgg 23
<210> 221
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 221
caagtgggag taaatggtgt agg 23
<210> 222
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 222
gttctgcaag tgggagtaaa tgg 23
<210> 223
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 223
tacacgggag ttctgcaagt ggg 23
<210> 224
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 224
gtacacggga gttctgcaag tgg 23
<210> 225
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 225
cagtaaagac acttgtacac ggg 23
<210> 226
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 226
gcagtaaaga cacttgtaca cgg 23
<210> 227
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 227
atctcatata aaaataaatg agg 23
<210> 228
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 228
cacacaccac aaacacaaac agg 23
<210> 229
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 229
tgcatgcctg tttgtgtttg tgg 23
<210> 230
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 230
ccctcaatat aaaccctttg tgg 23
<210> 231
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 231
ccacaaaggg tttatattga ggg 23
<210> 232
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 232
gccacaaagg gtttatattg agg 23
<210> 233
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 233
ttaaatatat gagccacaaa ggg 23
<210> 234
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 234
tttaaatata tgagccacaa agg 23
<210> 235
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 235
gattcagagc tttcaaggat agg 23
<210> 236
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 236
tccttgaaag ctctgaatca tgg 23
<210> 237
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 237
cccatgattc agagctttca agg 23
<210> 238
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 238
ccttgaaagc tctgaatcat ggg 23
<210> 239
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 239
atcatgggca gtgagctcag tgg 23
<210> 240
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 240
gcagtgagct cagtggtatc tgg 23
<210> 241
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 241
gtgagctcag tggtatctgg agg 23
<210> 242
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 242
ctcagtggta tctggaggac agg 23
<210> 243
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 243
tcagtggtat ctggaggaca ggg 23
<210> 244
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 244
gtatctggag gacagggcac tgg 23
<210> 245
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 245
gcagaagatg gtgactgcag tgg 23
<210> 246
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 246
tgcagtcacc atcttctgcc agg 23
<210> 247
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 247
gcaggcttcc tggcagaaga tgg 23
<210> 248
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 248
tgccaggaag cctgcacctc agg 23
<210> 249
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 249
gccaggaagc ctgcacctca ggg 23
<210> 250
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 250
cccctgaggt gcaggcttcc tgg 23
<210> 251
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 251
ccaggaagcc tgcacctcag ggg 23
<210> 252
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 252
agaattcacc cctgaggtgc agg 23
<210> 253
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 253
cggcaaagaa ttcacccctg agg 23
<210> 254
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 254
gggtgaattc tttgccgaaa tgg 23
<210> 255
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 255
gccgaaatgg attgccaaaa cgg 23
<210> 256
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 256
accgttttgg caatccattt cgg 23
<210> 257
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 257
aaatgtgctg gtgaccgttt tgg 23
<210> 258
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 258
cggtcaccag cacatttccc agg 23
<210> 259
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 259
acagctcctg ggaaatgtgc tgg 23
<210> 260
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 260
ctttcatctc aacagctcct ggg 23
<210> 261
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 261
ccaggagctg ttgagatgaa agg 23
<210> 262
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 262
cctttcatct caacagctcc tgg 23
<210> 263
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 263
tggagctctc agctcactat ggg 23
<210> 264
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 264
ctggagctct cagctcacta tgg 23
<210> 265
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 265
gtgagctgag agctccagcc tgg 23
<210> 266
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 266
tagttatctg gaggccaggc tgg 23
<210> 267
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 267
tgtgtagtta tctggaggcc agg 23
<210> 268
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 268
cttggtgtgt agttatctgg agg 23
<210> 269
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 269
aagcttggtg tgtagttatc tgg 23
<210> 270
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 270
cttgattctg ggtggaagct tgg 23
<210> 271
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 271
aacataggct tgattctggg tgg 23
<210> 272
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 272
agttaacata ggcttgattc tgg 23
<210> 273
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 273
ggcaaaatct caggctttga ggg 23
<210> 274
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 274
aggcaaaatc tcaggctttg agg 23
<210> 275
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 275
taatgggaag gcaaaatctc agg 23
<210> 276
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 276
tttgccttcc cattaaatgc agg 23
<210> 277
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 277
actacctgca tttaatggga agg 23
<210> 278
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 278
aacaactacc tgcatttaat ggg 23
<210> 279
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 279
gaacaactac ctgcatttaa tgg 23
<210> 280
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 280
ccagtgacta gtgcttgaag ggg 23
<210> 281
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 281
gccagtgact agtgcttgaa ggg 23
<210> 282
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 282
ggccagtgac tagtgcttga agg 23
<210> 283
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 283
agatagcaag atttaaatta tgg 23
<210> 284
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 284
acaacataca gggttcatgg tgg 23
<210> 285
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 285
ccatgaaccc tgtatgttgt agg 23
<210> 286
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 286
cctacaacat acagggttca tgg 23
<210> 287
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 287
tcttcagcct acaacataca ggg 23
<210> 288
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 288
aacaattgaa acatttgggc tgg 23
<210> 289
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 289
gaggaacaat tgaaacattt ggg 23
<210> 290
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 290
tttcaattgt tcctcacccc tgg 23
<210> 291
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 291
gcaaagtatg tccaggggtg agg 23
<210> 292
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 292
tgggggcaaa gtatgtccag ggg 23
<210> 293
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 293
atgggggcaa agtatgtcca ggg 23
<210> 294
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 294
gatgggggca aagtatgtcc agg 23
<210> 295
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 295
ggacatactt tgcccccatc tgg 23
<210> 296
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 296
ttgcccccat ctggaattaa agg 23
<210> 297
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 297
tatcctttaa ttccagatgg ggg 23
<210> 298
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 298
atatccttta attccagatg ggg 23
<210> 299
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 299
tatatccttt aattccagat ggg 23
<210> 300
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 300
ttatatcctt taattccaga tgg 23
<210> 301
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 301
tatatgtgtc cagctgatat agg 23
<210> 302
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 302
aaggctattc ctatatcagc tgg 23
<210> 303
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 303
tagccttagc aatgtatgtt tgg 23
<210> 304
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 304
tggccaaaca tacattgcta agg 23
<210> 305
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 305
gtcaaagtgg ggaactttgg tgg 23
<210> 306
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 306
tcagtcaaag tggggaactt tgg 23
<210> 307
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 307
atattggctc agtcaaagtg ggg 23
<210> 308
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 308
tatattggct cagtcaaagt ggg 23
<210> 309
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 309
atatattggc tcagtcaaag tgg 23
<210> 310
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 310
tgcaggcaga aggcatatat tgg 23
<210> 311
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 311
gttaaaaaga tgcaggcaga agg 23
<210> 312
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 312
tatggtcgtt aaaaagatgc agg 23
<210> 313
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 313
atctggaggc aggacaagta tgg 23
<210> 314
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 314
taaaacatct atctggaggc agg 23
<210> 315
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 315
gttttaaaac atctatctgg agg 23
<210> 316
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 316
gttgttttaa aacatctatc tgg 23
<210> 317
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 317
gttttaaaac aacaaaaatg agg 23
<210> 318
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 318
ttttaaaaca acaaaaatga ggg 23
<210> 319
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 319
aagatgaaag ttctttctac tgg 23
<210> 320
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 320
agaaaagaga ggtggaaatg agg 23
<210> 321
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 321
tgactttgag aaaagagagg tgg 23
<210> 322
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 322
ttttgacttt gagaaaagag agg 23
<210> 323
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 323
ttgtccatct agatttttag agg 23
<210> 324
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 324
agtgcctcta aaaatctaga tgg 23
<210> 325
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 325
gatttttaga ggcactcctt agg 23
<210> 326
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 326
tggtaatgtt ttagggccta agg 23
<210> 327
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 327
aggccctaaa acattaccac tgg 23
<210> 328
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 328
ggccctaaaa cattaccact ggg 23
<210> 329
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 329
gacccagtgg taatgtttta ggg 23
<210> 330
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 330
agacccagtg gtaatgtttt agg 23
<210> 331
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 331
ctaactgggc tgagacccag tgg 23
<210> 332
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 332
gaaactgcag aggactaact ggg 23
<210> 333
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 333
agaaactgca gaggactaac tgg 23
<210> 334
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 334
tgggggtgaa gaaactgcag agg 23
<210> 335
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 335
tttgaagata ctggggttgg ggg 23
<210> 336
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 336
gtttgaagat actggggttg ggg 23
<210> 337
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 337
tgtttgaaga tactggggtt ggg 23
<210> 338
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 338
ctgtttgaag atactggggt tgg 23
<210> 339
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 339
tgagctgttt gaagatactg ggg 23
<210> 340
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 340
gtgagctgtt tgaagatact ggg 23
<210> 341
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 341
tgtgagctgt ttgaagatac tgg 23
<210> 342
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 342
gtattgatct gagcacagca ggg 23
<210> 343
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 343
agtattgatc tgagcacagc agg 23
<210> 344
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 344
tctcgaggca acttagacaa cgg 23
<210> 345
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 345
ctaagttgcc tcgagactaa agg 23
<210> 346
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 346
gcctcgagac taaaggcaac agg 23
<210> 347
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 347
ccctgttgcc tttagtctcg agg 23
<210> 348
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 348
cctcgagact aaaggcaaca ggg 23
<210> 349
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 349
aacagggctg aaacatctcc tgg 23
<210> 350
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 350
gagaacttca aggtgagtcc agg 23
<210> 351
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 351
ggactcacct tgaagttctc agg 23
<210> 352
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 352
tgtggatcct gagaacttca agg 23
<210> 353
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 353
cacagtgcag ttcactcagc tgg 23
<210> 354
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 354
acagtgcagt tcactcagct ggg 23
<210> 355
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 355
aggtgccctt gagatcatcc agg 23
<210> 356
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 356
aagcacctgg atgatctcaa ggg 23
<210> 357
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 357
aaagcacctg gatgatctca agg 23
<210> 358
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 358
gagatcatcc aggtgctttg tgg 23
<210> 359
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 359
gggagatgcc acaaagcacc tgg 23
<210> 360
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 360
gtgctttgtg gcatctccca agg 23
<210> 361
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 361
aagaaggtgc tgacttcctt ggg 23
<210> 362
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 362
caagaaggtg ctgacttcct tgg 23
<210> 363
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 363
agtcaaggca catggcaaga agg 23
<210> 364
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 364
cttgccatgt gccttgactt tgg 23
<210> 365
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 365
ttgccatgtg ccttgacttt ggg 23
<210> 366
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 366
tgccatgtgc cttgactttg ggg 23
<210> 367
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 367
aaccccaaag tcaaggcaca tgg 23
<210> 368
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 368
catgggcaac cccaaagtca agg 23
<210> 369
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 369
gactttgggg ttgcccatga tgg 23
<210> 370
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 370
ggggttgccc atgatggcag agg 23
<210> 371
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 371
gcccatgatg gcagaggcag agg 23
<210> 372
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 372
tcctctgcct ctgccatcat ggg 23
<210> 373
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 373
gtcctctgcc tctgccatca tgg 23
<210> 374
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 374
tgatggcaga ggcagaggac agg 23
<210> 375
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 375
cagaggttct ttgacagctt tgg 23
<210> 376
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 376
caaagctgtc aaagaacctc tgg 23
<210> 377
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 377
aaagctgtca aagaacctct ggg 23
<210> 378
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 378
tcaaagaacc tctgggtcca tgg 23
<210> 379
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 379
caaagaacct ctgggtccat ggg 23
<210> 380
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 380
ttgtctaccc atggacccag agg 23
<210> 381
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 381
gggtccatgg gtagacaacc agg 23
<210> 382
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 382
ggctcctggt tgtctaccca tgg 23
<210> 383
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 383
ttgtcaatct cacaggctcc tgg 23
<210> 384
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 384
agaacagttt gacagtcaga agg 23
<210> 385
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 385
aggtcgcttc tcaggatttg tgg 23
<210> 386
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 386
acaaatcctg agaagcgacc tgg 23
<210> 387
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 387
aaaagtccag gtcgcttctc agg 23
<210> 388
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 388
cctgtgcctg gcaaaagtcc agg 23
<210> 389
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 389
cctggacttt tgccaggcac agg 23
<210> 390
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 390
ctggactttt gccaggcaca ggg 23
<210> 391
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 391
caggacaagg gagggaagga agg 23
<210> 392
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 392
cttccttccc tcccttgtcc tgg 23
<210> 393
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 393
tgaccaggac aagggaggga agg 23
<210> 394
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 394
ctggtgacca ggacaaggga ggg 23
<210> 395
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 395
tctggtgacc aggacaaggg agg 23
<210> 396
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 396
ggctctggtg accaggacaa ggg 23
<210> 397
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 397
aggctctggt gaccaggaca agg 23
<210> 398
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 398
gaaggtaggc tctggtgacc agg 23
<210> 399
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 399
tcaccagagc ctaccttccc agg 23
<210> 400
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 400
caccagagcc taccttccca ggg 23
<210> 401
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 401
aaccctggga aggtaggctc tgg 23
<210> 402
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 402
aggagaaacc ctgggaaggt agg 23
<210> 403
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 403
ctggaggaga aaccctggga agg 23
<210> 404
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 404
gatgctggag gagaaaccct ggg 23
<210> 405
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 405
agatgctgga ggagaaaccc tgg 23
<210> 406
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 406
gtgaatgtgg aagatgctgg agg 23
<210> 407
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 407
aaggtgaatg tggaagatgc tgg 23
<210> 408
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 408
cctgtggggc aaggtgaatg tgg 23
<210> 409
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 409
tatcacaagc ctgtggggca agg 23
<210> 410
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 410
gctactatca caagcctgtg ggg 23
<210> 411
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 411
ggctactatc acaagcctgt ggg 23
<210> 412
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 412
aggctactat cacaagcctg tgg 23
<210> 413
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 413
tcatttcaca gaggaggaca agg 23
<210> 414
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 414
catgggtcat ttcacagagg agg 23
<210> 415
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 415
ctcctctgtg aaatgaccca tgg 23
<210> 416
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 416
cgccatgggt catttcacag agg 23
<210> 417
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 417
gtgaaatgac ccatggcgtc tgg 23
<210> 418
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 418
tgacccatgg cgtctggact agg 23
<210> 419
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 419
gctcctagtc cagacgccat ggg 23
<210> 420
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 420
agctcctagt ccagacgcca tgg 23
<210> 421
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 421
actcgcttct ggaacgtctg agg 23
<210> 422
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 422
acgttccaga agcgagtgtg tgg 23
<210> 423
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 423
cagttccaca cactcgcttc tgg 23
<210> 424
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 424
cgagtgtgtg gaactgctga agg 23
<210> 425
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 425
gagtgtgtgg aactgctgaa ggg 23
<210> 426
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 426
ggctagggat gaagaataaa agg 23
<210> 427
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 427
cttcatccct agccagccgc cgg 23
<210> 428
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 428
caggggccgg cggctggcta ggg 23
<210> 429
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 429
ccaggggccg gcggctggct agg 23
<210> 430
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 430
cctagccagc cgccggcccc tgg 23
<210> 431
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 431
tgaggccagg ggccggcggc tgg 23
<210> 432
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 432
ccagtgaggc caggggccgg cgg 23
<210> 433
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 433
ccgccggccc ctggcctcac tgg 23
<210> 434
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 434
tatccagtga ggccaggggc cgg 23
<210> 435
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 435
agagtatcca gtgaggccag ggg 23
<210> 436
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 436
tagagtatcc agtgaggcca ggg 23
<210> 437
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 437
ttagagtatc cagtgaggcc agg 23
<210> 438
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 438
tagtcttaga gtatccagtg agg 23
<210> 439
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 439
actggatact ctaagactat tgg 23
<210> 440
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 440
tggtcaagtt tgccttgtca agg 23
<210> 441
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 441
gtttgccttg tcaaggctat tgg 23
<210> 442
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 442
cttgaccaat agccttgaca agg 23
<210> 443
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 443
caaggctatt ggtcaaggca agg 23
<210> 444
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 444
gctattggtc aaggcaaggc tgg 23
<210> 445
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 445
aaggctggcc aacccatggg tgg 23
<210> 446
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 446
cctggctaaa ctccacccat ggg 23
<210> 447
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 447
cccatgggtg gagtttagcc agg 23
<210> 448
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 448
ccctggctaa actccaccca tgg 23
<210> 449
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 449
ccatgggtgg agtttagcca ggg 23
<210> 450
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 450
tatctgtctg aaacggtccc tgg 23
<210> 451
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 451
atgcaaatat ctgtctgaaa cgg 23
<210> 452
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 452
tatttgcatt gagatagtgt ggg 23
<210> 453
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 453
atttgcattg agatagtgtg ggg 23
<210> 454
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 454
gcattgagat agtgtgggga agg 23
<210> 455
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 455
cattgagata gtgtggggaa ggg 23
<210> 456
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 456
attgagatag tgtggggaag ggg 23
<210> 457
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 457
aattagcagt atcctcttgg ggg 23
<210> 458
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 458
aaattagcag tatcctcttg ggg 23
<210> 459
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 459
aaaattagca gtatcctctt ggg 23
<210> 460
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 460
aaaaattagc agtatcctct tgg 23
<210> 461
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 461
actgaatcgg aacaaggcaa agg 23
<210> 462
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 462
ggaatgactg aatcggaaca agg 23
<210> 463
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 463
aaaaactgga atgactgaat cgg 23
<210> 464
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 464
agaataaatt agagaaaaac tgg 23
<210> 465
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 465
ggagaaggaa actagctaaa ggg 23
<210> 466
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 466
gggagaagga aactagctaa agg 23
<210> 467
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 467
gtttccttct cccatcatag agg 23
<210> 468
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 468
gtatcctcta tgatgggaga agg 23
<210> 469
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 469
ctcccatcat agaggatacc agg 23
<210> 470
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 470
gtcctggtat cctctatgat ggg 23
<210> 471
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 471
agtcctggta tcctctatga tgg 23
<210> 472
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 472
acggctgaca aaagaagtcc tgg 23
<210> 473
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 473
agagacaaga aggtaaaaaa cgg 23
<210> 474
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 474
cactggagct agagacaaga agg 23
<210> 475
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 475
tcttgtctct agctccagtg agg 23
<210> 476
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 476
ctttaaacta caggcctcac tgg 23
<210> 477
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 477
taccaatttt tgaaaagttc agg 23
<210> 478
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 478
accaattttt gaaaagttca ggg 23
<210> 479
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 479
tccctgaact tttcaaaaat tgg 23
<210> 480
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 480
gggattgtga aatgtgtttt agg 23
<210> 481
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 481
gtgaaatgtg ttttaggcat agg 23
<210> 482
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 482
tgtgttttag gcataggtcc agg 23
<210> 483
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 483
cataggtcca ggatttttga cgg 23
<210> 484
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 484
ataggtccag gatttttgac ggg 23
<210> 485
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 485
atttgtcccg tcaaaaatcc tgg 23
<210> 486
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 486
tagtctcttt cagttagcag tgg 23
<210> 487
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 487
tcagttagca gtggtttcta agg 23
<210> 488
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 488
attgagaaat taagagataa tgg 23
<210> 489
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 489
ctattactgc gctgaaactg tgg 23
<210> 490
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 490
tagatattga ggtaagcatt agg 23
<210> 491
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 491
gtacggataa gtagatattg agg 23
<210> 492
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 492
tgatttattt caaataggta cgg 23
<210> 493
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 493
agtcatgatt tatttcaaat agg 23
<210> 494
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 494
aaatatttga ttccatattc agg 23
<210> 495
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 495
tatacataca tacctgaata tgg 23
<210> 496
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 496
agtggtaaat acacatcatc tgg 23
<210> 497
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 497
agatgatgtg tatttaccac tgg 23
<210> 498
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 498
cagcacacac acttatccag tgg 23
<210> 499
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 499
cactggataa gtgtgtgtgc tgg 23
<210> 500
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 500
tgtgtgctgg ctgatgaccc agg 23
<210> 501
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 501
tggctgatga cccagggttt tgg 23
<210> 502
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 502
aagagctacg ccaaaaccct ggg 23
<210> 503
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 503
gaagagctac gccaaaaccc tgg 23
<210> 504
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 504
tctatgctca gtaaagatga tgg 23
<210> 505
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 505
gatgatggta gaatgttctt tgg 23
<210> 506
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 506
atggtagaat gttctttggc agg 23
<210> 507
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 507
atgttctttg gcaggtactg tgg 23
<210> 508
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 508
aattatcttg tataaatgct agg 23
<210> 509
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 509
aatgctaggt tcacttctca ggg 23
<210> 510
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 510
acataagatg tgcgtgtaca tgg 23
<210> 511
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 511
catggaaaac aactctaaag agg 23
<210> 512
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 512
aaaacaactc taaagaggca agg 23
<210> 513
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 513
aaacaactct aaagaggcaa ggg 23
<210> 514
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 514
attcgagtta taatagtgtg tgg 23
<210> 515
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 515
cagtgctgtt tctgacatat tgg 23
<210> 516
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 516
tgacatattg gaccattaac agg 23
<210> 517
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 517
gacatattgg accattaaca ggg 23
<210> 518
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 518
tattggacca ttaacagggt agg 23
<210> 519
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 519
aatacttcct accctgttaa tgg 23
<210> 520
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 520
agggtaggaa gtatttatgg tgg 23
<210> 521
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 521
aagtatttat ggtggttttt tgg 23
<210> 522
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 522
ttttggttct gttttgcttt tgg 23
<210> 523
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 523
tctagcaagg ttagagatca tgg 23
<210> 524
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 524
gattataatg ccagaagctc tgg 23
<210> 525
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 525
tgccagaagc tctggaattc tgg 23
<210> 526
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 526
agccagaatt ccagagcttc tgg 23
<210> 527
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 527
gctctggaat tctggcttat cgg 23
<210> 528
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 528
ctggaattct ggcttatcgg agg 23
<210> 529
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 529
agtttatccc ctaagctatc agg 23
<210> 530
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 530
caatcaacct gatagcttag ggg 23
<210> 531
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 531
tcaatcaacc tgatagctta ggg 23
<210> 532
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 532
ttcaatcaac ctgatagctt agg 23
<210> 533
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 533
gattgaaatt attataatat tgg 23
<210> 534
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 534
agctttacac aggatcatga agg 23
<210> 535
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 535
agcagatata agctttacac agg 23
<210> 536
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 536
tatctgctca ttgatacaga tgg 23
<210> 537
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 537
cattgataca gatggctaaa agg 23
<210> 538
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 538
tgggtaaatg tgtgtctttc tgg 23
<210> 539
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 539
gatgtgcagg cttgttacat ggg 23
<210> 540
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 540
ggtacatgtg caggatgtgc agg 23
<210> 541
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 541
aatttcgaag gtacatgtgc agg 23
<210> 542
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 542
tcatttcatt ttaatttcga agg 23
<210> 543
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 543
tgaaatgaaa taaaattaaa agg 23
<210> 544
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 544
ttaaaaggaa aaaaatgcca tgg 23
<210> 545
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 545
aaaaatgcca tggcaacatc agg 23
<210> 546
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 546
catcaggaag ttatctttta tgg 23
<210> 547
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 547
tatcttttat ggtctaaaaa tgg 23
<210> 548
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 548
atcttttatg gtctaaaaat ggg 23
<210> 549
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 549
tcaatagtat gctaaacaag ggg 23
<210> 550
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 550
ttcaatagta tgctaaacaa ggg 23
<210> 551
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 551
tttcaatagt atgctaaaca agg 23
<210> 552
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 552
tgaaaaataa caataaaaat cgg 23
<210> 553
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 553
agagtagacg caagggctac tgg 23
<210> 554
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 554
cataggcaga gtagacgcaa ggg 23
<210> 555
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 555
tcataggcag agtagacgca agg 23
<210> 556
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 556
taaatgaatg gctacttcat agg 23
<210> 557
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 557
aaattgaagg aataaatgaa tgg 23
<210> 558
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 558
acaagtttat aaaaaattga agg 23
<210> 559
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 559
ttgttttact aaaaaaaaaa agg 23
<210> 560
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 560
agaaagtgcg aattgtgaaa tgg 23
<210> 561
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 561
tgtgaaatgg tagtgagtga tgg 23
<210> 562
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 562
gtgagtgatg gcatttgaag tgg 23
<210> 563
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 563
tgagtgatgg catttgaagt ggg 23
<210> 564
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 564
gtgggtcctt tatgatttga tgg 23
<210> 565
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 565
ctggctccat caaatcataa agg 23
<210> 566
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 566
ggagccaggc agaagacgtt tgg 23
<210> 567
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 567
agaaagaagt tcctgaaagt agg 23
<210> 568
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 568
agaagttcct gaaagtagga agg 23
<210> 569
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 569
gaagttcctg aaagtaggaa ggg 23
<210> 570
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 570
cacatgccct tcctactttc agg 23
<210> 571
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 571
ctgaaagtag gaagggcatg tgg 23
<210> 572
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 572
agggcatgtg gaaaactctg agg 23
<210> 573
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 573
tgtggaaaac tctgaggctg agg 23
<210> 574
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 574
ttttttcttc aacgtacttt agg 23
<210> 575
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 575
agaattcaaa gttagacaga agg 23
<210> 576
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 576
gaattcaaag ttagacagaa ggg 23
<210> 577
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 577
aaagttagac agaagggctc agg 23
<210> 578
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 578
acagatgaga gtagtagagt tgg 23
<210> 579
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 579
tattttattc tgaaacacag agg 23
<210> 580
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 580
tctgaaacac agaggacaag tgg 23
<210> 581
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 581
ttagaaacca tgtttatctc agg 23
<210> 582
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 582
aaataagctt cggtgttcag tgg 23
<210> 583
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 583
aaagagaaaa aaataagctt cgg 23
<210> 584
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 584
atagcacctt gcactaagta agg 23
<210> 585
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 585
tataacaaaa tagcatagac tgg 23
<210> 586
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 586
ataacaaaat agcatagact ggg 23
<210> 587
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 587
gactgggtga cttcaacaac agg 23
<210> 588
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 588
attttttctc acttttctgc tgg 23
<210> 589
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 589
ctcacttttc tgctggttcc tgg 23
<210> 590
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 590
ttttctgctg gttcctggtc agg 23
<210> 591
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 591
tttctgctgg ttcctggtca ggg 23
<210> 592
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 592
ccaggaagag agccctgacc agg 23
<210> 593
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 593
cctggtcagg gctctcttcc tgg 23
<210> 594
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 594
ctggtcaggg ctctcttcct ggg 23
<210> 595
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 595
gggctctctt cctgggttgc agg 23
<210> 596
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 596
ctctcttcct gggttgcagg tgg 23
<210> 597
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 597
aagtcagcca cctgcaaccc agg 23
<210> 598
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 598
ccttctggtg aggacacagt ggg 23
<210> 599
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 599
cccactgtgt cctcaccaga agg 23
<210> 600
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 600
tccttctggt gaggacacag tgg 23
<210> 601
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 601
catttctctt ccttctggtg agg 23
<210> 602
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 602
ctcaccagaa ggaagagaaa tgg 23
<210> 603
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 603
ttagccattt ctcttccttc tgg 23
<210> 604
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 604
taatctctct gcttctgatg agg 23
<210> 605
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 605
tgaggaaaat aattctatga tgg 23
<210> 606
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 606
gaggaaaata attctatgat ggg 23
<210> 607
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 607
aggaaaataa ttctatgatg ggg 23
<210> 608
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 608
ggaaaagtaa tcaggtttag agg 23
<210> 609
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 609
gaggctttgg aaaagtaatc agg 23
<210> 610
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 610
tccaaagcct cccccacaaa tgg 23
<210> 611
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 611
tccatttgtg ggggaggctt tgg 23
<210> 612
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 612
attggctcca tttgtggggg agg 23
<210> 613
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 613
cccccacaaa tggagccaat tgg 23
<210> 614
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 614
ccaattggct ccatttgtgg ggg 23
<210> 615
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 615
cccaattggc tccatttgtg ggg 23
<210> 616
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 616
ccccacaaat ggagccaatt ggg 23
<210> 617
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 617
tcccaattgg ctccatttgt ggg 23
<210> 618
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 618
ctcccaattg gctccatttg tgg 23
<210> 619
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 619
aaatggagcc aattgggagt tgg 23
<210> 620
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 620
aatggagcca attgggagtt ggg 23
<210> 621
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 621
atggagccaa ttgggagttg ggg 23
<210> 622
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 622
ctgaatcccc aactcccaat tgg 23
<210> 623
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 623
gattcagcat gtgaattttc tgg 23
<210> 624
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 624
aagagtttag tttaatgtga tgg 23
<210> 625
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 625
aattgagaag gcagattcac tgg 23
<210> 626
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 626
ttgtataaca ttaattgaga agg 23
<210> 627
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 627
gtctcagcag cagaacagca cgg 23
<210> 628
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 628
gctgacttgt gagcttctgc tgg 23
<210> 629
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 629
ggaggtgggg acttaggtga agg 23
<210> 630
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 630
ttctagggag gtggggactt agg 23
<210> 631
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 631
tgtaagcttc tagggaggtg ggg 23
<210> 632
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 632
ttgtaagctt ctagggaggt ggg 23
<210> 633
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 633
attgtaagct tctagggagg tgg 23
<210> 634
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 634
ctcattgtaa gcttctaggg agg 23
<210> 635
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 635
ctccctagaa gcttacaatg agg 23
<210> 636
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 636
gacctcattg taagcttcta ggg 23
<210> 637
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 637
ctggtcacag catttcaagg agg 23
<210> 638
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 638
agactggtca cagcatttca agg 23
<210> 639
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 639
gtgaccagtc tgtagactta agg 23
<210> 640
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 640
tgaccagtct gtagacttaa ggg 23
<210> 641
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 641
agtctgtaga cttaagggta tgg 23
<210> 642
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 642
gtctgtagac ttaagggtat ggg 23
<210> 643
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 643
gggattaatt tcacctcttt agg 23
<210> 644
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 644
gtgttgacgc atgcctaaag agg 23
<210> 645
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 645
aagtgatcct tactttacta agg 23
<210> 646
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 646
tcacttcatt gtagttaccg tgg 23
<210> 647
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 647
gaaacaggtt tttctttcca cgg 23
<210> 648
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 648
tagagatcag agcaggaaac agg 23
<210> 649
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 649
gaggtgttag agatcagagc agg 23
<210> 650
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 650
gaggctctac agggaggttg agg 23
<210> 651
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 651
ccttgtgagg ctctacaggg agg 23
<210> 652
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 652
cctccctgta gagcctcaca agg 23
<210> 653
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 653
ctccctgtag agcctcacaa ggg 23
<210> 654
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 654
agcccttgtg aggctctaca ggg 23
<210> 655
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 655
cagcccttgt gaggctctac agg 23
<210> 656
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 656
ggcttttctc agcccttgtg agg 23
<210> 657
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 657
tgagaaaagc cagatgccgt tgg 23
<210> 658
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 658
atactatttc caacggcatc tgg 23
<210> 659
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 659
aggggcaata ctatttccaa cgg 23
<210> 660
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 660
ttgcccctaa taaaatagaa tgg 23
<210> 661
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 661
tagccattct attttattag ggg 23
<210> 662
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 662
atagccattc tattttatta ggg 23
<210> 663
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 663
gatagccatt ctattttatt agg 23
<210> 664
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 664
aggaagtcaa ggtttaggca ggg 23
<210> 665
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 665
taggaagtca aggtttaggc agg 23
<210> 666
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 666
gctttaggaa gtcaaggttt agg 23
<210> 667
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 667
aaccttgact tcctaaagct tgg 23
<210> 668
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 668
ttccaagctt taggaagtca agg 23
<210> 669
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 669
gaaaaagtgt tccaagcttt agg 23
<210> 670
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 670
gcagcacagc agcgaggaag ggg 23
<210> 671
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 671
ttgcagcaca gcagcgagga agg 23
<210> 672
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 672
tggattgcag cacagcagcg agg 23
<210> 673
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 673
tcgctgctgt gctgcaatcc agg 23
<210> 674
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 674
ctgtgctgca atccagggaa ggg 23
<210> 675
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 675
tgtgctgcaa tccagggaag ggg 23
<210> 676
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 676
gggggctgaa cccccttccc tgg 23
<210> 677
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 677
ggggaaaggt tcagtgaagg ggg 23
<210> 678
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 678
tggggaaagg ttcagtgaag ggg 23
<210> 679
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 679
gatggggaaa ggttcagtga agg 23
<210> 680
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 680
gttttggaag agatggggaa agg 23
<210> 681
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 681
cagatgtttt ggaagagatg ggg 23
<210> 682
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 682
ttccaaaaca tctgtttctg agg 23
<210> 683
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 683
ctcctcagaa acagatgttt tgg 23
<210> 684
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 684
tgaggagtcc tgtcctatag agg 23
<210> 685
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 685
gttggaagaa agacctctat agg 23
<210> 686
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 686
aggtgcagga gaaatctggt tgg 23
<210> 687
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 687
taaaaggtgc aggagaaatc tgg 23
<210> 688
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 688
caagtgggag taaaaggtgc agg 23
<210> 689
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 689
tacatgggag ttctgcaagt ggg 23
<210> 690
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 690
gtacatggga gttctgcaag tgg 23
<210> 691
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 691
caataaagaa acttgtacat ggg 23
<210> 692
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 692
gcaataaaga aacttgtaca tgg 23
<210> 693
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 693
gatgagcaaa taaaagaact agg 23
<210> 694
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 694
tttttgttta cgttagatgt ggg 23
<210> 695
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 695
ttttttgttt acgttagatg tgg 23
<210> 696
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 696
ctaacgtaaa caaaaaagtg tgg 23
<210> 697
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 697
tgtttatgtg tatttataca tgg 23
<210> 698
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 698
ccacaaaggg ttttatgttg agg 23
<210> 699
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 699
cctcaacata aaaccctttg tgg 23
<210> 700
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 700
ttctgctaag agatcacaca tgg 23
<210> 701
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 701
gatgcagagc tttcaaggat agg 23
<210> 702
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 702
tccttgaaag ctctgcatca tgg 23
<210> 703
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 703
cccatgatgc agagctttca agg 23
<210> 704
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 704
ccttgaaagc tctgcatcat ggg 23
<210> 705
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 705
gcagaagatg gtgactggag tgg 23
<210> 706
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 706
tcctggcaga agatggtgac tgg 23
<210> 707
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 707
agcaaaatct caggctttga ggg 23
<210> 708
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 708
aagcaaaatc tcaggctttg agg 23
<210> 709
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 709
taatgggaaa gcaaaatctc agg 23
<210> 710
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 710
tttgctttcc cattaaatgc agg 23
<210> 711
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 711
cttcttgcag cactagtcac tgg 23
<210> 712
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 712
agataacaag atttaaatta tgg 23
<210> 713
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 713
acagcataca gggttcatgg tgg 23
<210> 714
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 714
cctacagcat acagggttca tgg 23
<210> 715
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 715
ccatgaaccc tgtatgctgt agg 23
<210> 716
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 716
ttttcagcct acagcataca ggg 23
<210> 717
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 717
gttttcagcc tacagcatac agg 23
<210> 718
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 718
attaaaaaga tgcaggcaga agg 23
<210> 719
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 719
tatggtcatt aaaaagatgc agg 23
<210> 720
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 720
ttcgttttaa aacatctatc tgg 23
<210> 721
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 721
ttaaaacgaa taacaaaaat agg 23
<210> 722
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 722
taaaacgaat aacaaaaata ggg 23
<210> 723
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 723
aaaacgaata acaaaaatag ggg 23
<210> 724
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 724
gaataacaaa aataggggaa agg 23
<210> 725
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 725
gacttttctt tattagattt cgg 23
<210> 726
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 726
ctttgacttt gagaaaagag agg 23
<210> 727
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 727
ttgtccatct agattttcag agg 23
<210> 728
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 728
agtgcctctg aaaatctaga tgg 23
<210> 729
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 729
gattttcaga ggcactcctt agg 23
<210> 730
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 730
tggcaatgtt ttagggccta agg 23
<210> 731
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 731
aggccctaaa acattgccac tgg 23
<210> 732
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 732
ggccctaaaa cattgccact ggg 23
<210> 733
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 733
gacccagtgg caatgtttta ggg 23
<210> 734
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 734
agacccagtg gcaatgtttt agg 23
<210> 735
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 735
tgggagtgaa gaaactgcag agg 23
<210> 736
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 736
cactgttgcc tttagtctcg agg 23
<210> 737
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 737
aacagtgctg aaacatctcc tgg 23
<210> 738
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 738
gagatcatcc aggtgcttta tgg 23
<210> 739
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 739
gggagatgcc ataaagcacc tgg 23
<210> 740
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 740
gtgctttatg gcatctccca agg 23
<210> 741
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 741
aagcagcagt atcctcttgg ggg 23
<210> 742
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 742
taagcagcag tatcctcttg ggg 23
<210> 743
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 743
ttaagcagca gtatcctctt ggg 23
<210> 744
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 744
aaaaattgga atgactgaat cgg 23
<210> 745
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 745
agaataaatt agagaaaaat tgg 23
<210> 746
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 746
ggagaagaaa actagctaaa ggg 23
<210> 747
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 747
gggagaagaa aactagctaa agg 23
<210> 748
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 748
ctcccaccat agaagatacc agg 23
<210> 749
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 749
gtcctggtat cttctatggt ggg 23
<210> 750
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 750
agtcctggta tcttctatgg tgg 23
<210> 751
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 751
agaagtcctg gtatcttcta tgg 23
<210> 752
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 752
acagacaaga aggtgaaaaa cgg 23
<210> 753
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 753
tcttgtctgt agctccagtg agg 23
<210> 754
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 754
atgctttaac tttaaactac agg 23
<210> 755
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 755
acgtgtccca tcaaaaatcc tgg 23
<210> 756
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 756
tagtctcatt tagtaagcat tgg 23
<210> 757
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 757
taagtatagc acttcttatt tgg 23
<210> 758
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 758
atctttgaat atactctctg tgg 23
<210> 759
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 759
aatatactct ctgtggtttt agg 23
<210> 760
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 760
aatgagaact taagagataa tgg 23
<210> 761
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 761
cactgaaact gttgctttat agg 23
<210> 762
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 762
taaagcaaca gtttcagtgc agg 23
<210> 763
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 763
gataagtaga tattgaagta agg 23
<210> 764
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 764
aggcgtgatt tatttcaaat agg 23
<210> 765
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 765
tacttttgct gagatgaaac agg 23
<210> 766
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 766
aaagtatttt ttttatattc agg 23
<210> 767
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 767
ttatattcag gtatgtatgt agg 23
<210> 768
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 768
agtgataaat acacatcatc ggg 23
<210> 769
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 769
cagtgataaa tacacatcat cgg 23
<210> 770
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 770
cgatgatgtg tatttatcac tgg 23
<210> 771
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 771
cactggataa gtgtatgtgc tgg 23
<210> 772
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 772
atgtgctggc tgatgaccca ggg 23
<210> 773
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 773
aagagctaca ccaaaaccct ggg 23
<210> 774
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 774
gaagagctac accaaaaccc tgg 23
<210> 775
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 775
tggtgtagct cttctatgct cgg 23
<210> 776
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 776
tctatgctcg gtaaagatga tgg 23
<210> 777
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 777
atgctcttta ccagatacta tgg 23
<210> 778
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 778
taattctaat ccatagtatc tgg 23
<210> 779
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 779
aaatgctagg ttcacttctt agg 23
<210> 780
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 780
aatgctaggt tcacttctta ggg 23
<210> 781
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 781
gaaaacaact cttaagaggc agg 23
<210> 782
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 782
aaaacaactc ttaagaggca ggg 23
<210> 783
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 783
agtccacaca ctgtgacagc tgg 23
<210> 784
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 784
gtccacacac tgtgacagct ggg 23
<210> 785
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 785
atcccagctg tcacagtgtg tgg 23
<210> 786
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 786
gacatattgg ccacttaaca tgg 23
<210> 787
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 787
tattggccac ttaacatggc agg 23
<210> 788
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 788
atacttcctg ccatgttaag tgg 23
<210> 789
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 789
ttttttcttt attttttctt tgg 23
<210> 790
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 790
tttttcttta ttttttcttt ggg 23
<210> 791
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 791
tctttgggat ttttattttt tgg 23
<210> 792
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 792
tgggattttt attttttggt agg 23
<210> 793
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 793
tttttatttt ttggtaggtt tgg 23
<210> 794
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 794
ctgaactcga tccatgacct tgg 23
<210> 795
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 795
gtcataatct accaaggtca tgg 23
<210> 796
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 796
tctgacgtca taatctacca agg 23
<210> 797
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 797
gattatgacg tcagaaactc tgg 23
<210> 798
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 798
cgtcagaaac tctggaaatc tgg 23
<210> 799
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 799
actctggaaa tctggcttat tgg 23
<210> 800
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 800
ctggaaatct ggcttattgg agg 23
<210> 801
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 801
ctaccttccc ctaacctatc agg 23
<210> 802
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 802
caacctgata ggttagggga agg 23
<210> 803
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 803
ctatcaacct gataggttag ggg 23
<210> 804
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 804
cctatcaacc tgataggtta ggg 23
<210> 805
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 805
ccctaaccta tcaggttgat agg 23
<210> 806
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 806
tcctatcaac ctgataggtt agg 23
<210> 807
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 807
ataattccta tcaacctgat agg 23
<210> 808
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 808
gataggaatt ataataatat tgg 23
<210> 809
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 809
aggaattata ataatattgg tgg 23
<210> 810
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 810
acaggattat gaagtctgaa agg 23
<210> 811
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 811
agacttcata atcctgtgta agg 23
<210> 812
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 812
agcagatata agccttacac agg 23
<210> 813
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 813
tatctgctca aagtgttagg cgg 23
<210> 814
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 814
caggtcaaca tgtatctttc tgg 23
<210> 815
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 815
tatgtgcagc tttgttgcgc agg 23
<210> 816
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 816
cacatattgc acttgttctt tgg 23
<210> 817
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 817
aaataaaagt tgaataagaa agg 23
<210> 818
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 818
cttattatta tgtgagtaac tgg 23
<210> 819
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 819
aagtgacttc tctcaatcta agg 23
<210> 820
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 820
gacttctctc aatctaagga tgg 23
<210> 821
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 821
acttctctca atctaaggat ggg 23
<210> 822
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 822
tgggtgagaa agatttttat tgg 23
<210> 823
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 823
aaagattttt attggtatgc tgg 23
<210> 824
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 824
ggtacagatt atagagactg agg 23
<210> 825
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 825
gactgaggtg aacccaaatt agg 23
<210> 826
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 826
actgaggtga acccaaatta ggg 23
<210> 827
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 827
tcttaaagag accctaattt ggg 23
<210> 828
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 828
atcttaaaga gaccctaatt tgg 23
<210> 829
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 829
aattagggtc tctttaagat agg 23
<210> 830
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 830
attagggtct ctttaagata ggg 23
<210> 831
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 831
ttagggtctc tttaagatag ggg 23
<210> 832
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 832
aagatagggg atctgtgaga tgg 23
<210> 833
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 833
agatagggga tctgtgagat ggg 23
<210> 834
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 834
gatgggtgtg tatagatctt tgg 23
<210> 835
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 835
tttggccata tctgagagtc tgg 23
<210> 836
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 836
gccatatctg agagtctggt tgg 23
<210> 837
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 837
cccaaccaga ctctcagata tgg 23
<210> 838
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 838
ccatatctga gagtctggtt ggg 23
<210> 839
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 839
taaaggtcag gctctacaaa tgg 23
<210> 840
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 840
ctgacacatt cttaaaggtc agg 23
<210> 841
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 841
agtctctgac acattcttaa agg 23
<210> 842
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 842
tttaagaatg tgtcagagac tgg 23
<210> 843
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 843
actggtgtct aagcccgcta agg 23
<210> 844
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 844
tcacttgttt aggccttagc ggg 23
<210> 845
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 845
ttcacttgtt taggccttag cgg 23
<210> 846
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 846
ttcaaaagat tcacttgttt agg 23
<210> 847
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 847
agagtcttgt tctgtcaccc agg 23
<210> 848
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 848
tcttgttctg tcacccaggc tgg 23
<210> 849
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 849
ataccactgc actccagcct ggg 23
<210> 850
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 850
cataccactg cactccagcc tgg 23
<210> 851
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 851
cactgcaact cctgccttcc agg 23
<210> 852
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 852
attgcttgaa cctggaaggc agg 23
<210> 853
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 853
gagaattgct tgaacctgga agg 23
<210> 854
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 854
tcctcagcct cccaagtagc tgg 23
<210> 855
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 855
cccagctact tgggaggctg agg 23
<210> 856
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 856
cctcagcctc ccaagtagct ggg 23
<210> 857
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 857
tgtaatccca gctacttggg agg 23
<210> 858
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 858
tcccaagtag ctgggattac agg 23
<210> 859
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 859
gcctgtaatc ccagctactt ggg 23
<210> 860
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 860
tgcctgtaat cccagctact tgg 23
<210> 861
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 861
caaaaattag acaggcatga tgg 23
<210> 862
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 862
caaaaataca aaaattagac agg 23
<210> 863
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 863
tttttgtatt tttgtagaga tgg 23
<210> 864
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 864
ttttgtattt ttgtagagat ggg 23
<210> 865
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 865
gagatgggat ttcaccatgt tgg 23
<210> 866
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 866
gggatttcac catgttggcc agg 23
<210> 867
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 867
tttcaccatg ttggccaggt tgg 23
<210> 868
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 868
caagaccaac ctggccaaca tgg 23
<210> 869
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 869
tcaagagttc aagaccaacc tgg 23
<210> 870
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 870
ggtcttgaac tcttgacctc agg 23
<210> 871
<211> 1605
<212> PRT
<213> Artificial Sequence
<220>
<223> ABE8e
<400> 871
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala
85 90 95
Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys
115 120 125
Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1010 1015 1020
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1025 1030 1035
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1040 1045 1050
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1055 1060 1065
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1070 1075 1080
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1085 1090 1095
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1100 1105 1110
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1115 1120 1125
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1130 1135 1140
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1145 1150 1155
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1160 1165 1170
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1175 1180 1185
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1190 1195 1200
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1205 1210 1215
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1220 1225 1230
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1250 1255 1260
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1265 1270 1275
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1280 1285 1290
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1295 1300 1305
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1310 1315 1320
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1325 1330 1335
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1340 1345 1350
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1355 1360 1365
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1370 1375 1380
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1385 1390 1395
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1400 1405 1410
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1415 1420 1425
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1430 1435 1440
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1445 1450 1455
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1460 1465 1470
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1490 1495 1500
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1505 1510 1515
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1520 1525 1530
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1535 1540 1545
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1550 1555 1560
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1565 1570 1575
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp
1580 1585 1590
Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1595 1600 1605
<210> 872
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> AAVS1 sgRNA
<400> 872
tttatcacag gctccaggaa 20
<210> 873
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA1620
<400> 873
ctccctccca ggatcctctc 20

Claims (10)

1.一种靶向HBG启动子的sgRNA文库，其包含如SEQ ID NO:1、2和3所示的核苷酸序列中的一种或多种。
2.如权利要求1所述的sgRNA文库，其特征在于，所述sgRNA文库还包含如SEQ ID NO:4-SEQ ID NO:59所示的核苷酸序列中的一种或多种；
较佳地，所述sgRNA文库进一步包含如SEQ ID NO:59-SEQ ID NO:870所示的核苷酸序列中的一种或多种；
更佳地，所述sgRNA位于病毒载体上，所述病毒载体优选为慢病毒载体。
3.一种碱基编辑系统，其包含ABE8e碱基编辑器和如权利要求1或2所述的sgRNA文库；较佳地，所述ABE8e碱基编辑器的序列如SEQ ID NO:871所示。
4.一种药物组合物，其特征在于，所述药物组合物包括如权利要求3所述的碱基编辑系统；较佳地，所述药物组合物还包括药物递送系统；更佳地，所述药物递送系统为脂质体，外泌体、噬菌体或细菌微细胞。
5.一种基因工程细胞，其特征在于，所述基因工程细胞包含如权利要求3所述的碱基编辑系统。
6.如权利要求5所述的基因工程细胞，其特征在于，所述基因工程细胞的出发细胞为造血干细胞、造血祖细胞或红系祖细胞；和/或，所述出发细胞的来源为哺乳动物；
较佳地，所述出发细胞为CD34+造血干细胞或CD34+造血祖细胞；和/或，所述出发细胞的来源为人或鼠；例如所述出发细胞为人HUDEP-2细胞。
7.一种非治疗目的的碱基编辑方法，其特征在于，所述碱基编辑方法通过如权利要求3所述的碱基编辑系统、如权利要求4所述的药物组合物、或者如权利要求5或6所述的基因工程细胞对HBG启动子进行碱基编辑。
8.一种非治疗目的的提高HbF表达的方法，其包括使用如权利要求7所述的碱基编辑方法；优选地，所述提高HbF表达通过提高γ珠蛋白RNA表达实现。
9.如权利要求1或2所述的sgRNA文库、权利要求3所述的碱基编辑系统、权利要求4所述的药物组合物、或者如权利要求5或6所述的基因工程细胞在制备碱基编辑的药物中的应用；
较佳地，所述碱基编辑的药物为治疗遗传性疾病的药物；
更佳地，所述遗传性疾病为地中海贫血；
进一步更佳地，所述遗传性疾病为β地中海贫血。
10.如权利要求1或2所述的sgRNA文库、权利要求3所述的碱基编辑系统、权利要求4所述的药物组合物、或者如权利要求5或6所述的基因工程细胞在制备碱基编辑工具或碱基编辑模型中的应用。
CN202111123978.3A 2021-09-24 2021-09-24 一种碱基编辑系统及其应用 Pending CN115851706A (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202111123978.3A CN115851706A (zh) 2021-09-24 2021-09-24 一种碱基编辑系统及其应用
PCT/CN2022/120949 WO2023046086A1 (zh) 2021-09-24 2022-09-23 一种碱基编辑系统及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111123978.3A CN115851706A (zh) 2021-09-24 2021-09-24 一种碱基编辑系统及其应用

Publications (1)

Publication Number Publication Date
CN115851706A true CN115851706A (zh) 2023-03-28

Family

ID=85652664

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111123978.3A Pending CN115851706A (zh) 2021-09-24 2021-09-24 一种碱基编辑系统及其应用

Country Status (2)

Country Link
CN (1) CN115851706A (zh)
WO (1) WO2023046086A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116790593B (zh) * 2023-06-28 2024-05-10 贵州医科大学 一种激活γ珠蛋白表达的sgRNA及其CRISPR/Cas9复合体与应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019236209A1 (en) * 2018-03-14 2020-10-01 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN109385425A (zh) * 2018-11-13 2019-02-26 中山大学 一种高特异性ABE碱基编辑系统及其在β血红蛋白病中的应用
CN112011576A (zh) * 2019-05-31 2020-12-01 华东师范大学 Crispr基因编辑技术在治疗地中海贫血中的应用
CN111876416B (zh) * 2020-07-01 2021-09-03 广州瑞风生物科技有限公司 激活γ-珠蛋白基因表达的方法和组合物

Also Published As

Publication number Publication date
WO2023046086A1 (zh) 2023-03-30

Similar Documents

Publication Publication Date Title
US11963982B2 (en) CRISPR/RNA-guided nuclease systems and methods
US11851690B2 (en) Systems and methods for the treatment of hemoglobinopathies
US20210254061A1 (en) Systems and methods for the treatment of hemoglobinopathies
CN111712569A (zh) 用于基因编辑的Cpf1-相关方法和组合物
US20220073951A1 (en) Systems and methods for the treatment of hemoglobinopathies
JP6066356B2 (ja) 改善された遺伝子治療方法
US20210230638A1 (en) Systems and methods for the treatment of hemoglobinopathies
US20200263206A1 (en) Targeted integration systems and methods for the treatment of hemoglobinopathies
CN111718420A (zh) 一种用于基因治疗的融合蛋白及其应用
CN115851706A (zh) 一种碱基编辑系统及其应用
US20220047637A1 (en) Systems and methods for the treatment of hemoglobinopathies
US20240335476A1 (en) Systems and methods for the treatment of hemoglobinopathies
CN118056014A (zh) 单碱基编辑修复hba2基因突变的方法及其应用
CN115873850A (zh) 腺嘌呤碱基编辑系统及其应用
WO2022143694A1 (zh) 一种针对细胞中单个或多个基因进行基因编辑的方法、产品和应用
WO2022234051A1 (en) Split prime editing enzyme
WO2023212657A2 (en) Enhancement of safety and precision for crispr-cas induced gene editing by variants of dna polymerase using cas-plus variants
WO2024123842A1 (en) Systems and methods for the treatment of hemoglobinopathies
JP2016129524A (ja) 改善された遺伝子治療方法

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination