Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.
Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.
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DOI：
10.1186/s13100-022-00279-x
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发表时间：
2022-11-10
期刊：
影响因子：
4.9
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中科院分区：
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Krüppel相关盒锌指蛋白（KRAB-ZFP）是哺乳动物中最大的转录因子超家族，主要靶向和抑制转座因子（TE）。由于这些转录调节子的序列相似性和多样性，以及它们的靶向TE序列的复杂重复性，剖析它们的不同功能是具有挑战性的。小鼠KRAB-Zfps主要在基因组范围内组织成簇。在这项研究中，我们发现，簇内成员有一个密切的进化关系，和类似的偏好锌指（ZnF）的使用。KRAB-Zfps以细胞类型或组织类型特异性的方式表达，并且它们倾向于与其他簇成员一起活跃地转录。进一步的序列分析表明，ZnFs之间的连接子序列是保守的，同时具有明显的簇特异性。基于KRAB-Zfp簇的这些独特特征，设计sgRNA以编辑簇特异性接头以消除靶向簇的功能。以小鼠胚胎干细胞（mESC）为模型，我们筛选并获得了一系列靶向各种高表达KRAB-Zfp簇的sgRNA。sgRNA的有效性在专门为多靶sgRNA开发的报告基因测定中得到验证，并通过基于PCR的分析进一步证实。使用诱导表达Cas9和这些sgRNA的mESC细胞系，我们发现编辑不同的KRAB-Zfp簇导致不同类别的TE的转录变化。总的来说，本研究中发现的簇内KRAB-Zfp成员的内在序列相关性表明，保守的簇特异性接头在簇进化过程中使串联ZnF阵列多样化和KRAB-Zfp的相关靶特异性方面发挥了关键作用。在此基础上，开发并验证了针对接头序列的有效的基于CRISPR-Cas9的方法，用于快速编辑KRAB-Zfp簇，以鉴定簇成员与其潜在TE靶标之间的调控相关性。在线版本包含补充材料，可通过10.1186/s13100-022-00279-x获得。
Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences. Mouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs. Collectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters’ evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets. The online version contains supplementary material available at 10.1186/s13100-022-00279-x.
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