Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression

Abstract

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

Genome editing methods in animal models

“…The Takai group generated a mouse model of ACD and CD by deleting the cartilage-specific SOX9 enhancer, termed the rib-cage-specific enhancer, using the embryo microinjection method of the CRISPR system. This model was consistent with the clinical phenotypes of human ACD/CD patients and is expected to be applicable to studies aimed at the treatment of ACD/CD (Mochizuki et al 2018).…”

Insights into Gene Regulatory Networks in Chondrocytes

“…The effect of the enhancer perturbation on putative target gene expressions was assessed by profiling 254,974 single-cell transcriptomes, resulting in the identification of 470 highly confident enhancer-gene pairs. Moreover, Mochizuki et al conducted a combinatorial assay of CRISPR screening with mass spectrometry, focusing on Sox9 gene regulatory elements [65]. They identified a chondrocyte-specific Sox9 enhancer that was activated by Stat3 [65].…”

“…Moreover, Mochizuki et al conducted a combinatorial assay of CRISPR screening with mass spectrometry, focusing on Sox9 gene regulatory elements [65]. They identified a chondrocyte-specific Sox9 enhancer that was activated by Stat3 [65]. These screening technologies are powerful tools in the field and will be extensively used with higher throughput.…”

Enhancing CRISPR deletion via pharmacological delay of DNA-PK

“…In the case of precise genome editing, which is based on the HDR pathway, efficiency has been substantially improved through pharmacological promotion of HDR and inhibition of the competing NHEJ pathway 37 . No such solutions have been developed for CRISPR-del, despite its being one of the most common CRISPR-Cas9 modalities, with diverse scientific and technological applications [5][6][7][8][9][10][11][12][13][14][15] .…”

“…One such application, CRISPR-deletion (CRISPR-del), is a means of permanently removing specific genomic fragments from 10 1 -10 6 base pairs 4 . This range has enabled researchers to investigate a wide variety of functional elements, including gene regulatory sequences [5][6][7] , non-coding RNAs [8][9][10][11][12] , and structural elements 13 . Similarly, engineered deletions can be used to model human mutations 14,15 .…”