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

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.