Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9
Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9
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DOI：
10.4161/rna.29624
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发表时间：
2014-07-01
期刊：
影响因子：
4.1
通讯作者：
Zhang, Wensheng
中科院分区：
文献类型：
--
作者：
Han, Jinxiong;Zhang, Jun;Zhang, Wensheng
最近的全基因组研究表明，大多数小鼠基因组转录为非编码RNAs(NcRNAs)，越来越多的证据支持ncRNAs在调控基因表达和表观遗传过程中的重要性。然而，传统的基因打靶策略的低效率阻碍了体内ncRNAs的功能研究，特别是在产生具有多种表达变体的长非编码RNAs(LncRNAs)的大片段缺失方面。细菌簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关9(Cas9)系统最近已被用作设计基因组蛋白质编码基因位点特异性突变的有效工具。在这项研究中，我们探索了使用CRISPR/Cas9系统在小鼠中产生大量基因组缺失的lncRNAs的可能性。我们开发了一种有效的一步策略来靶向在小鼠12号染色体上母体表达的lncRNA，Rian。我们发现成对的sgRNA可以精确地产生高达23kb的大片段缺失，通过组合多个sgRNA可以进一步提高删除效率高达33%。该缺失成功地取消了Rian在母系遗传等位基因中的表达，验证了突变在研究印记基因座中的生物学相关性。Rian突变对邻近基因在不同的体细胞组织中的表达有不同的影响。综上所述，我们已经建立了一种强大的一步法，利用CRISPR/Cas9系统工程大量缺失的lncRNA基因。我们的工作将为将来在体内进行其他lncRNA的功能研究提供便利。
Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.