Kennedy Outlaw, a junior studying biochemistry (Chemistry) and applied physics at Purdue University, prime edited blood cancer mutations in spliceosome protein SF3B1.
Splice factor 3B unit 1 (SF3B1) is an essential gene involved in splicing that is necessary for survival. Splicing is a highly regulated cellular process required to generate protein diversity and cell type specific protein expression that is necessary for complex eukaryotic organisms.The Broad at face value is already an incredible environment where collaboration and cutting edge research merge beautifully. The much more meaningful part of the Broad that I also had the privilege of experiencing are the amazing connections and motivation between peers, mentors, and everyone you could possibly come across. 95% of the genes in our genome undergo alternative splicing, which is regulated by the spliceosome and other RNA binding proteins. SF3B1 plays an important role in splicing by recognizing the 3’ splice site on the intron-exon junction. Splicing factor mutations primarily occur in hematologic malignancies, with SF3B1 being the most commonly mutated. Hotspot mutations, such as SF3B1 K700E and K666N, arise at the pre-mRNA and SF3B1 interface and disrupt the interaction of SF3B1 with the 3’ splice site leading to mis-splicing of a subset of transcripts. To further investigate why K700E and K666N contribute to a phenotype of cancer, we will utilize CRISPR prime editing to endogenously introduce these mutations into CD34+ hematopoietic stem cells to model splicing factor alteration in a relevant cell context. To this end, we created a panel of pegRNAs of differing lengths of primer binding sequence and reverse transcriptase template to determine the optimal features for best efficiency of prime editing. We found the highest prime editing efficiency with K700E targeting pegRNAs with the use of an intermediate length primer binding sequence and the shortest reverse transcriptase template, and will need to further optimize reagents for K666N.
Project: Using prime editing to model splicing factor mutations in the spliceosome protein SF3B1 in blood cancers
Mentor: Mounica Vallurupalli, MD, Golub Lab, Cancer Program