TitleGenome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.
Publication TypeJournal Article
Year of Publication2022
AuthorsDevaraju N, Rajendiran V, Ravi NSam, Mohankumar KM
JournalMethods Mol Biol
Date Published2022
KeywordsAnimals, CRISPR-Cas Systems, Gene Editing, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Mice, Transplantation, Autologous

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.

Alternate JournalMethods Mol Biol
PubMed ID35507170


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    Guide RNAs are designed to target specific DNA sequences, inducing breaks. Natural repair mechanisms either create mutations (NHEJ) or incorporate desired changes (HDR). Edited HSCs are screened and transplanted back into the patient, potentially correcting genetic anomalies. Challenges include off-target effects, efficient delivery, and safety. Ongoing research aims to refine this method for effective and secure genome editing in HSCs. https://answersville.com/.

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