一种CRISPR-Cas9介导的拟南芥高效基因编辑系统的构建与应用

Development of an Efficient Editing System in Arabidopsis by CRISPR-Cas9

【背景】近些年兴起的CRISPR-Cas9基因编辑技术在多种植物中实现了高效的基因打靶,为基因功能研究提供了一种高效快速的方法,但一些CRISPR-Cas9载体编辑效率很低。【目的】通过构建一种由RIBOSOMAL PROTEIN S5 A(RPS5A)启动子启动Cas9并带有红色荧光蛋白筛选标记的CRISPR-Cas9载体,提高拟南芥CRISPR-Cas9编辑效率,并利用这套系统对拟南芥木葡聚糖内糖基转移/水解酶基因TOUCH4(TCH4)进行编辑。【方法】在pKSE401载体的基础上,以从胚胎发育早期就表现出高转录活性的RPS5A启动子替换35S启动子、以DsRed2替换潮霉素抗性基因,构建拟南芥中使用的CRISPR载体pRSE-WH;以AtTCH4为靶基因,使用CRISPR-P 2.0(http://crispr.hzau.edu.cn)设计靶位点,将所设计的靶点序列在拟南芥参考基因组中比对分析以排除非特异性靶位点,最终筛选出2个靶位点target 1和target 2。化学合成带有接头的靶位点寡核苷酸序列,退火后分别与pRSE-WH载体连接,构建TCR1和TCR2表达载体,采用农杆菌介导的沾花法侵染野生型拟南芥Col-0,以红色荧光蛋白为标记筛选获得T1代转基因阳性植株。通过靶位点扩增测序法判断T1代转基因植株在预期靶位点是否发生编辑,根据测序结果的峰图对编辑情况进行解码,进一步分析突变类型及基因型。【结果】构建了一个在拟南芥中高效编辑的CRISPR载体pRSE-WH。TCR1和TCR2成功地实现了对TCH4的定点编辑,靶点一的编辑效率为80%,靶点二的编辑效率为100%,总编辑效率为86%。根据测序结果的峰图解码了T1代植株的突变结果,纯合编辑、杂合编辑、双等位编辑均有出现。对不同的编辑类型进行统计发现,59株T1代阳性植株中,无编辑8株,占比13.56%,纯合编辑9株,占比15.25%,双等位编辑40株,占比67.80%,杂合编辑2株,占比3.39%。在T1代发生纯合编辑以及双等位编辑的株系中选择了无红光种子进行繁种,并对T2代植株编辑情况进行测序检测,结果发现T1代中的突变成功遗传到了T2代无Cas9株系中。【结论】pRSE-WH在拟南芥中展现了极高的编辑效率,并且通过对种子进行红色荧光筛选,能够简便地获得无Cas9且稳定遗传的T3代突变体。

【Background】In recent years,the CRISPR-Cas9 system has been developed to an efficient site-directed geneediting technology in a variety of plants.It can provide a rapid method for studying gene function.However,the editing efficiency of some CRISPR-Cas9 vectors is very low.【Objective】In this study,an efficient CRISPR-Cas9 system,with a red fluorescent protein screening marker and a RIBOSOMAL PROTEIN S5 A(RPS5A)promoter,was constructed to edit the xyloglucan endotransglucosylase/hydrolase TOUCH4(TCH4)gene in Arabidopsis thaliana.【Method】The CRISPR vector pRSE-WH was constructed from the pKSE401 vector by replacing the 35S promoter with a RPS5A promoter,which maintains high constitutive expression at all developmental stages from the egg cell,and the hygromycin resistance gene with DsRed2.The sgRNA of TCH4 was designed using CRISPR-P 2.0(http://crispr.hzau.edu.cn).After excluding non-specific target sites by sequence comparisons with the Arabidopsis thaliana genome database,two target sites were selected.The sgRNA oligonucleotides with junctions were chemically synthesized.After annealing,two expression vectors(TCR1 and TCR2,corresponding to the two target sites)were constructed by linking the sgRNA with junctions to the plasmid pRSE-WH respectively.The vectors of TCR1 and TCR2 were transferred into wild-type Col-0 by Agrobacterium-mediated floral-dip transformation.In the T1 generation,the positive transgenic plants were identified using red fluorescent protein as a marker,and the target sites were detected using PCR combined with Sanger-sequencing to confirm whether it was mutated as expected.The mutations were determined from the peak map of the sequencing results,and the mutation pattern and genotypes were further analyzed.【Result】A high efficiency CRISPR vector pRSE-WH for gene editing in Arabidopsis was constructed.The TCR1 and TCR2 expression vectors successfully enabled gene-specific editing of TCH4.In the editing of the TCH4 gene,the mutation ratios using TCR1 and TCR2 were 80%,100%respectively,and the total editing efficiency was 86%.The mutations in T1 plants were decoded according to the sequencing results and included homozygous,heterozygous and biallelic mutations.Among 59 mutated T1 plants,8 plants(13.56%)were not edited,9 plants(15.25%)were homozygous,40 plants(67.80%)were biallelic and 2 plants(3.39%)were heterozygous.The homozygous and biallelic seeds without red fluorescence of T1 generation were selected for breeding,and sequenced the plants of T2 generation.The results showed that mutations of the T1 generation were successfully inherited to T2 generation,which were Cas9-free.【Conclusion】PRSE-WH demonstrated high-efficiency gene editing in Arabidopsis thaliana.It was very easy to obtain Cas9-free and stably inherited mutants in the T3 generation by screening seeds with red fluorescence marker.