CRISPR-Cas9 aracılığı ile kromatin modifiye eden genlerin insan hücre yeniden programlanmasında incelenmesi

Reprogramming of somatic cells to pluripotency via four transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM) involves extensive remodeling of the epigenome which is carried out by a number of chromatin modifiers. A number of these genes have been studied in the context of reprogramming and defined as barriers or essential regulators of this process. However, established protocols limit a comprehensive examination due to the susceptibility of shRNA vectors to proviral silencing which accompanies reprogramming, and the unavailability of human knock-out cell lines. Therefore, we hypothesized that pooled CRISPR-Cas9 knock-out libraries can enable systematic investigation of chromatin modifiers and reveal their role in different steps of reprogramming by enabling permanent gene modifications. To this end, we constructed a CRISPR-Cas9 knock-out library targeting the 247 chromatin modifiers including chromatin remodelers, writers, readers and erasers of histone and DNA modifications. We utilized this library to generate heterogeneous cell populations harboring putative chromatin modifier knock-outs, which were then reprogrammed with the delivery of OSKM. FACS-based enrichment of TRA-1-60 positive cells, and low copy exogenous retroviral EGFP silencing was utilized to collect emerging iPSCs at different time points of reprogramming. The abundance of CRISPR guide RNAs in isolated cells were determined by next generation sequencing. Analysis of relative enrichment of specific guide RNAs confirmed the majority of the known reprogramming regulators and identified novel stage specific barriers and essential regulators for human somatic cell reprogramming. These include PAXIP1, BRD2 and USP22 as barriers, and CHAF1A, KDM8 and PRMT5 as essential regulators. In addition, functional analysis based on class of genes revealed that various MLL complexes act as barriers, and BAD, SWI/SNF, and MOZ/MORF complexes act as essential regulators for reprogramming. Based on the screen results, stage specific combinatorial use of small molecule inhibitors enabled accelerated and more synchronous efficient reprogramming. Taken together this study establishes roadblocks of reprogramming through chromatin landscape, and can guide more efficient reprogramming strategies to be formulated.