Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response

Abstract

CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9–gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.

Prostaglandin E2 Enhances Aged Hematopoietic Stem Cell Function

“…Cebpb is the gene for CCAAT enhancer binding protein beta (C/EBPβ), a transcription factor that promotes lymphopoiesis 65 and emergency myelopoiesis 66,67 at the level of stem and progenitor cell regulation 68,69 . Gadd45b, encoding growth arrest and DNA-damage-inducible beta (GADD45β), appears essential for DNA damage protection and survival of HSCs/HPCs and induced pluripotent stem cells (iPSCs) under stress 70 . Cdkn1a encodes the cyclin-dependent kinase inhibitor P21, which preserves HSC quiescence under stress and promotes HSC self-renewal in serial transplantation 71 , while Nr4a2 encoding the transcription factor nuclear receptor subfamily 4 group A member 2, also known as NURR1, also attenuates HSC cycling 72 and may contribute to the maintenance of stem cell quiescence during the stress of transplantation.…”

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients

“…We recently reported the establishment of the fluorescent labeling of CRISPR/Cas9 RNP complexes for gene editing of primary hematopoietic stem cells and subsequent sorting of gene-modified cells for further applications. 26 Implementation of this method will improve the efficiency of gene knockout or gene correction in HSPC, including ELANE knockout or correction of ELANE mutations.…”

Prostaglandin E2 Enhances Aged Hematopoietic Stem Cell Function

“…Many of these genes additionally have described roles in hematopoiesis, supporting their involvement in HSC modulation by dmPGE 2 . Vegfa encodes for vascular endothelial growth factor A (VEGF-A) which, while rst discovered for its primary role in angiogenesis 60 70 . Cdkn1a encodes the cyclin-dependent kinase inhibitor P21, which preserves HSC quiescence under stress and promotes HSC self-renewal in serial transplantation 71 , while Nr4a2 encoding the transcription factor nuclear receptor subfamily 4 group A member 2, also known as NURR1, also attenuates HSC cycling 72 and may contribute to the maintenance of stem cell quiescence during the stress of transplantation.…”