Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Abstract

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesisregulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Ppm1d Promotes Inflammation and Non-Ischemic Heart Failure in Mice

“…To establish the Ppm1d clonal hematopoiesis model, a lentivirus-mediated, CRISPR-Cas9 approach was employed to induce mutations in Ppm1d in bone marrow cells before transplantation into recipient mice. 16 , 29 The PPM1D mutations commonly associated with clonal hematopoiesis are restricted to exon 6 that will promote protein stability and increase in phosphatase activity. 20 , 23 , 24 Thus, bone marrow cells from transgenic mice expressing Cas9 and EGFP from the Rosa26 locus were transfected with lentivirus encoding the TagRFP (Tag red fluorescent protein) reporter gene and a single guide RNA (sgRNA) that targets one of 2 regions in exon 6 of the Ppm1d gene (Figure 1 A).…”

Hematopoietic loss of Y chromosome leads to cardiac fibrosis and heart failure mortality

“…1A). To decouple the Cas9 gene from the lentivirus vector and to maximize gene-editing efficiency, donor bone marrow cells were isolated from ROSA26-Cas9 knock-in mice that express the Cas9 endonuclease systemically ( 11 ). Two LOY-gRNAs, LOY-gRNA1 and LOY-gRNA2, targeting different repeat sequences within the centromere were evaluated for the efficiency of Y chromosome ablation by fluorescent in situ hybridization (FISH) analysis of X and Y chromosomes in tRFP + blood cells collected from mice reconstituted with bone marrow cells.…”

JAK2-Mediated Clonal Hematopoiesis Accelerates Pathological Remodeling in Murine Heart Failure

“…psPAX2 and pMD2.G were a gift from Didier Trono (Addgene, Watertown, Massachusetts, plasmids 12260 and 12259). Lentivirus particles were generated as described previously (34). Briefly, the plasmids (pLenti-SP146-gp91-JAK2, psPAX2, pMD2.G) were co-transfected to HEK293T cells with polyethylenimine (Cat# 24765-1, Polysciences, Warrington, Pennsylvania) and the supernatant was collected at 48 h after transfection.…”