CRISPR-BASED EPIGENETICS: A NEW ERA IN DEVELOPMENTAL BIOLOGY

Abstract

 The CRISPR based epigenetic engineering has proved quite precise and programmable to adapt in the expression of genes without altering the sequence of the genes themselves. This creates new avenues of studying developmental regulatory networks.  Coupled catalytically dead dCas9 together with transcriptional effectors such as KRAB and p300 was used in this study to target promoter, enhancer and CpG-rich regions in pluripotent and embryonic stem cells.  Alterations in the expression of guided epigenetic changes involved change of histone marks (like H3 K27 ac and H3 K9 me 3) and DNA methylation in specific loci that altered the expression of genes in a large extent.  The RNA-Seq and qRT-PCR assays revealed transcriptional repression of the KRAB-modified regions and strongly activated p300-fused constructs with a measured increase in the expression levels of up to three-fold in certain sites.  Time-course analysis had revealed temporal spikes and prolonged effects of regulative activity post-editing.  The integrated omics analysis used implied the presence of an intense correlation between chromatin state and transcriptional output (r > 0.8). Phenotypic assays, however, showed that development trajectories have shifted, like shifting spheroid shape and lineage marker markings.  Bioinformatic modelling indicated that we can predict about the gene expression on the basis of epigenetic characteristics. This further renders this platform more agreeable in management of development.  The study in sum reveals that CRISPR-based epigenetic editing is a powerful and modulatable method to pose queries about and manipulate cell fate. This leaves the alternatives open to new thinking in the areas of developmental biology, regenerative and synthetic embryology.