Gene ( IF 3.5 ) Pub Date : 2020-07-21 , DOI: 10.1016/j.gene.2020.144975 Zin Mar Oo 1 , Salah Adlat 1 , Rajiv Kumar Sah 1 , May Zun Zaw Myint 1 , Farooq Hayel 1 , Yang Chen 1 , Hsu Htoo 1 , Fatoumata Binta Bah 1 , Noor Bahadar 1 , Mi Kaythi Chan 2 , Luqing Zhang 3 , Xuechao Feng 4 , Yaowu Zheng 4
Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including ‘memory’, ‘neuropeptide signaling pathway’, and ‘response to amphetamine’ while KEGG analysis shows that ‘neuroactive ligand-receptor interaction pathway’ is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.
中文翻译:
通过CRISPR / Cas9介导的小鼠Dip2c基因敲除进行脑转录组研究。
Dip2C在脑和许多其他组织中高度表达,但其生物学功能仍不清楚。从未研究过大脑中由Dip2C调控的基因。最近已经开发出簇状的规则间隔的短回文重复序列(CRISPR)/ CRISPR相关蛋白(Cas)系统,细菌和古细菌的适应性免疫系统,并广泛用于基因组编辑中。在这里,我们描述了通过CRISPR / Cas9系统在小鼠中Dip2c基因的靶向基因缺失以及在Dip2C调控下的脑转录组研究。CRISPR / Cas9系统有效地产生了Dip2c的靶向缺失通过前核注射表达Cas9蛋白和两个sgRNA的质粒。我们在3个独立的实验中分别实现了14.3%(1/7),66.7%(2/3)和20%(1/5)的靶向大片段缺失,平均效率为26.7%。的大缺失的DNA区段是160.4千字节(DIP2C Δ160kb),跨越从外显子4的端部具有两个碱基对的缺失外显子38,一种鼠标的中间从单个因组中的外显子4(靶向产生DIP2C Δ2bp通过非同源)结束加入(NHEJ)。通过定量实时PCR(qPCR)证实了Dip2c mRNA的基因表达缺失。DIP2C调节基因和途径在大脑通过RNA测序进行了调查DIP2C Δ2bp。总共发现了838个基因的差异调控,其中252个向上和586个向下。基因本体论(GO)分析表明,大脑中的DEG富含神经功能,包括“记忆”,“神经肽信号传导途径”和“对苯丙胺的反应”,而KEGG分析表明,“神经活性配体-受体相互作用途径”是最显着的丰富。DEG Grid2ip,Grin2a,Grin2c,Grm4,Gabbr2,Gabra5,Gabre,Gabrq,Gabra6和Gabrr2是Dip2C高度调控的基因。结果证实Dip2C可能在大脑发育和功能中起重要作用。