目的:探讨microRNA-140（miR-140）是否有独立于其宿主基因WWP2的转录启动子，从而为骨性关节炎的治疗提供新的思路。方法:通过共转染CRISPR-dCas9-VP64和ACAN gRNA慢病毒的SW1353细胞验证软骨细胞特征性基因ACAN的转录水平。通过构建能稳定表达CRISPR-dCas9-VP64的SW1353细胞并结合多条sgRNA（single guide RNA, sgRNA/gRNA）、人类髋关节软骨RNA测序结果验证miR-140是否与WWP2基因共表达，是否有独立的转录启动位点。结果:共转染CRISPR-dCas9-VP64和ACAN gRNA慢病毒的SW1353细胞能显著提高软骨细胞特征性基因ACAN的转录水平。从基因水平、蛋白水平证实成功构建能稳定表达CRISPR-dCas9-VP64的SW1353细胞。通过人类髋关节软骨RNA测序结果设计多条WWP2 gRNA以及miR-140启动子gRNA。实时定量PCR结果显示miR-140的表达既受其宿主基因WWP2调控，同时也有其独立的转录位点。结论:运用CRISPR-Cas9基因编辑技术成功构建稳定表达CRISPR-dCas9-VP64蛋白的SW1353细胞能极大减少实验中样本间的差异化，保证实验结果的一致性。结合多条gRNA证明了miR-140既与WWP2共表达，同时也受其独立的转录启动子调控转录。运用CRISPR-dCas9-VP64d-gRNA能单独调控miR-140的表达，同时其宿主基因的表达不受影响，有潜力作为新兴的基因治疗方法治疗骨性关节炎。

The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineer-ing and transcriptional regulation in many different organisms.Current CRISPR-activation(CRISPRa)platforms often require multi-ple components because of inefficient transcriptional activation.Here,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation efficiency.Notably,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity.dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR.These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants.We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR-associated protein 9 (dCas9) fused with four tandem repeats of the transcriptional activator VP16 (VP64)could be used for transcriptional activation of endogenous genes in plants.In this study,we developed a second generation of vector systems for enhanced transcriptional activation in plants.We tested multiple strategies for dCas9-based transcriptional activation,and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than the dCas9-VP64 system.Moreover,we developed a multiplex transcription activatorlike effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants.Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested.In addition,we explored tissue-specific gene activation using positive feedback loops.Interestingly,our study revealed that certain endogenous genes are more amenable than others to transcriptional activation,and tightly regulated genes may cause target gene silencing when perturbed by activation probes.Hence,these new tools could be used to investigate gene regulatory networks and their control mechanisms.Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies,which will facilitate transcriptional activation applications in both dicots and monocots.

Dear Editor,The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system is an adaptive immune system in a variety of bacteria and archaea (Terns and Terns,2011).The most commonly used Streptococcus pyogenes type Ⅱ CRISPR-Cas9 system consists of Cas9 nuclease and two short RNAs,crRNA and tracrRNA,which can be linked together forming one chimeric single guide RNA (sgRNA) (Jinek et al.,2012).Guided by sgRNA,Cas9-sgRNA complex can generate DNA double strand breaks (DSB) at specific genomic loci (Jinek et al.,2012;Cong et al.,2013;Mali et al.,2013).Cas9 with mutations at the catalytic RuvC and HNH domains loses the endonuclease activity (indicated "dCas9"),while maintains its DNA binding capability (Gilbert et al.,2013).DCas9 fused with transcription effectors,such as VP64,P65-HSF1 and KRAB can be guided to specific promoters by sgRNA,enabling regulation of gene transcription (Gilbert et al.,2013,2014).

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms.We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR fromPrevotella andFrancisella 1 (Cpf1) could induce target mutations,deletions,inversions,and duplications both singly and multiplex in silkworm,Bombyx mori.However,it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B.mori.In this study,we investigated the CRISPRa system,in which a nuclease dead Streptococcuspyogenes Cas9 (SpCas9) is fused to two transcription activation domains,including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain),and VPR (a tripartite activator,composed of VP64,p65,and Rta).The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B.mori cells,of which the latter showed significantly higher activity.The dCas9-VPR system showed considerable activity on all five tested target genes,and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level.We also observed that this system could be used to upregulate a range of target genes.Taken together,our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B.mori and perhaps other non-drosophila insects.