Whole-genome Epigenetic Function Annotation Through SgRNA Libraries Synthesized By Controlled Templatedependent Elongation

Epigenome is the set of DNA or DNA-associated proteins of chemical modifications,which regulates gene expression in processes of development and disease without changing DNA sequence.While current advances have allowed researchers to routinely profile epigenomes from given samples,our understandings of the functions of epigenetic hallmarks are nonspecific at best.Applying CRISPR-screening to genome-widely interrogate the function of individual epigenetic hallmarks demands massive sgRNA libraries which are unaffordable via commercial syntheses.We have developed a high-throughput,simple and cost-effective controlled template-dependent elongation(CTDE)approach that can convert any DNA sample to a sgRNAs library,which covers 98.47% of the effective CRISPR/Cas9 targeting sites within the source DNA.Significantly,over 99% CTDE-sgRNAs targeting sequences have a protospacer adjacent motif(PAM).Based on CTDE approach,several breakthroughs have ensued.We have generated sgRNA libraries targeting all H3K4me3 and CTCF hallmarks in mouse embryonic stem cells(mESCs)and all H3K4me3 hallmarks in HepG2.In total,we have screened 2.8M sgRNAs and after filtered ssgRNAs via CFD scores,we identified 12K(12887)H3K4me3 and 2K(2206)CTCF essential hallmarks for the proliferation of mESCs and HepG2,which render the first batch of functional epigenome annotation of H3K4me3 and CTCF hallmarks in mammalian cells.mESCs CTCF dataset shows that mESCs maintain a high proportion of non-essential cell-type specific CTCF hallmarks,which may be important for the implement of pluripotency.Various findings through characterizing essential H3K4me3 hallmark in HepG2 have shown the significance of functional epigenome annotation in cancer research.