CRISPR/Cas9 genome-wide loss of function screening identifies novel regulators of reprogramming to pluripotency

In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability \nof four transcription factors; Oct4, Sox2, Klf4 and c-Myc to ‘reprogram’ differentiated \nsomatic cells to a pluripotent state. This technology holds huge potential in the field \nof regenerative medicine, but reprogramming also a model system by which to the \ncommon regulators of all forced cell identity changes, for example, \ntransdifferentiation. Despite this, the mechanism underlying reprogramming remains \npoorly understood and the efficiency of induced pluripotent stem cell (iPSC) \ngeneration, inefficient. One powerful method for elucidating the gene components \ninfluencing a biological process, such as reprogramming, is screening for a phenotype \nof interest using genome-wide mutant libraries. Historically, large-scale knockout \nscreens have been challenging to perform in diploid mammalian genomes, while other \nscreening technologies such as RNAi can be disadvantaged by variable knockdown of \ntarget transcripts and off-target effects. \n \nComponents of clustered regularly interspaced short palindromic repeats and \nassociated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have \nrecently been adapted to edit genomic sequences at high efficiency in mammalian \nsystems. Furthermore, the application of CRISPR-Cas components to perform proofof- \nprinciple genome-wide KO screens has been successfully demonstrated. I have \nutilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening \nin the context of murine iPSC reprogramming, identifying 18 novel inhibitors of \nreprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and \nGtf2i. Understanding how these novel reprogramming roadblocks function to inhibit \nthe reprogramming process will provide insight into the molecular mechanisms \nunderpinning forced cell identity changes.