RNA-sequencing of Zbtb33 CRISPR edited mouse LSK cells

The goal of this project was to evaluate the impact of Zbtb33 loss on alternative splicing and gene expression in mouse hematopoietic stem and progenitor cells (HSPCs). We used CRISPR/Cas9 editing in male HSPCs to introduce loss of function mutations into the single Zbtb33 allele in Cas9 transgenic mice. Competitive transplants were performed in which lethally irradiated recipient mice (n=5) were transplanted with a 1:1 mix of LSKs (Lin-Sca+Kit+ bone marrow) lentivirally transduced with a sgRNA targeting Zbtb33 or a control locus (an intronic region in Gapdh). We utilized lentiviral sgRNA plasmids that also strongly express tagRFP (Zbtb33) or tagBFP (control). 38 weeks post transplant, bone marrow was harvested, and RNA was extracted from RFP+ (Zbtb33 sgRNA positive) and RFP- (Zbtb33 sgRNA negative) FACS-sorted LSKs.