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

Abstract

Background 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. Results 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. Conclusions 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.

Systematic single-cell analysis reveals dynamic control of transposable element activity orchestrating the endothelial-to-hematopoietic transition

“…The majority of TE silencers were downregulated in pre-HECs compared to VECs and HSCs (for example, 84.07 and 77.49% of KRAB-ZFPs in human and mouse, respectively) (Fig. 4 C, F; Additional file 8 : Table S5 and S6; KRAB-ZFP genes are available from [ 66 ]). Among the downregulated KRAB-ZFPs, ZNF84, ZNF382, and ZNF429 were found to bind significantly to L1 superfamily in human [ 67 ].…”

“…Genes related to TE silencing were collected from the literature [ 41 , 43 ]. Potential KRAB-ZFP genes in human and mouse were obtained from [ 66 ]. The whole list of TE silencers analyzed in this study can be found in Additional file 8 : Table S5 and S6.…”

Systematic single-cell analysis reveals dynamic control of transposable element activity orchestrating the endothelial-to-hematopoietic transition

“…Surprisingly, the majority of TE silencers were downregulated in pre-HECs against VECs and HSCs (for example, 84.07% and 77.49% of KRAB-ZFPs in human and mouse, respectively) (Fig. 4C, F; Additional file 7: Table S9 and S10; KRAB-ZFP genes are available from [63]). Among the downregulated KRAB-ZPFs, ZNF84, ZNF382 and ZNF429 were found to bind significantly to L1 superfamily in human [64].…”

“…Genes related to TE silencing were collected from the literature [42,44]. Potential KRAB-ZFP genes in human and mouse were obtained from [63]. The whole list of TE silencers analyzed in this study can be found in Additional file 7: Table S9 and S10.…”