CAJ | 학술논문

CRISPR/Cas9系统介导的基因组编辑技术是新一代功能强大的基因修饰技术。然而，脱靶效应(Off-target effects)是目前CRISPR/Cas9技术面临的最大问题。因此，设计脱靶风险低的sgRNA(single guide RNA)就成为关键。sgRNAcas9是一款专门用于sgRNA设计和评估脱靶效应的软件包。针对其核心运行程序，我们利用Java程序语言开发了其图形用户界面。此外，依据脱靶位点碱基数和 sgRNA 种子序列(seed sequences)的特异性，通过设置不同的风险等级对sgRNA的脱靶效应进行评估。随后，利用此软件设计了34124条靶向人、小鼠、大鼠、猪和鸡中共4691个microRNA(miRNA)前体的sgRNAs。此外，随机挑选了一个靶向人miR-206前体的sgRNA进行脱靶效应评估和验证。结果发现，sgRNAcas9软件人机交互界面友好，大多数miRNA前体可通过该软件寻找到 sgRNA，且他们的 GC%含量范围集中于40%~60%。利用 sgRNAcas9软件设计的靶向 miR-206的 sgRNA，其基因组编辑活性和脱靶位点可被实验验证。本研究表明 sgRNAcas9图形用户界面软件能针对任意物种设计特异性的sgRNA，其可在BiooTools(http：//www.biootools.com/)网站下载。
CRISPR/Cas9계통개도적기인조편집기술시신일대공능강대적기인수식기술。연이，탈파효응(Off-target effects)시목전CRISPR/Cas9기술면림적최대문제。인차，설계탈파풍험저적sgRNA(single guide RNA)취성위관건。sgRNAcas9시일관전문용우sgRNA설계화평고탈파효응적연건포。침대기핵심운행정서，아문이용Java정서어언개발료기도형용호계면。차외，의거탈파위점감기수화 sgRNA 충자서렬(seed sequences)적특이성，통과설치불동적풍험등급대sgRNA적탈파효응진행평고。수후，이용차연건설계료34124조파향인、소서、대서、저화계중공4691개microRNA(miRNA)전체적sgRNAs。차외，수궤도선료일개파향인miR-206전체적sgRNA진행탈파효응평고화험증。결과발현，sgRNAcas9연건인궤교호계면우호，대다수miRNA전체가통과해연건심조도 sgRNA，차타문적 GC%함량범위집중우40%~60%。이용 sgRNAcas9연건설계적파향 miR-206적 sgRNA，기기인조편집활성화탈파위점가피실험험증。본연구표명 sgRNAcas9도형용호계면연건능침대임의물충설계특이성적sgRNA，기가재BiooTools(http：//www.biootools.com/)망참하재。
The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, off-target effects of the Cas9 nuclease activity is a recurrent concern of the CRISPR system. Thus, designing sgRNA (single guide RNA) with minimal off-target effects is very important. sgRNAcas9 is a software package, which can be used to design sgRNA and to evaluate potential off-target cleavage sites. In this study, a graphical user interface for sgRNAcas9 was developed using the Java programming language. In addition, off-target effect for sgRNAs was eva-luated according to mismatched number and“seed sequence”specification. Moreover, sgRNAcas9 software was used to design 34 124 sgRNAs, which can target 4691 microRNA (miRNA) precursors from human, mouse, rat, pig, and chicken. In particular, the off-target effect of a sgRNA targeting to human miR-206 precursor was analyzed, and the on/off-target activity of this sgRNA was validated by T7E1 assay in vitro. Taken together, these data showed that the interface can simplify the usage of the sgRNAcas9 program, which can be used to design sgRNAs for the majority of miRNA precursors. We also found that the GC% of those sgRNAs ranged from 40% to 60%. In summary, the sgRNAcas9 software can be easily used to design sgRNA with minimal off-target effects for any species. The soft-ware can be downloaded from BiooTools website (http://www.biootools.com/).