TY - JOUR T1 - Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System. JF - Methods Mol Biol Y1 - 2022 A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Ravi, Nithin Sam A1 - Mohankumar, Kumarasamypet M KW - Animals KW - CRISPR-Cas Systems KW - Gene Editing KW - Hematopoietic Stem Cell Transplantation KW - Hematopoietic Stem Cells KW - Mice KW - Transplantation, Autologous AB -

Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.

VL - 2429 ER -