Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem and progenitor cells

TL;DR

This paper provides a detailed protocol for efficiently editing the genome of human blood stem cells using CRISPR and AAV, while preserving cell function.

Contribution

A novel, efficient protocol for CRISPR/AAV-mediated genome editing in hematopoietic stem cells with preserved viability and engraftment.

Findings

A protocol for AAV production, purification, and genome editing in HSPCs is described.

High editing efficiency is achieved while maintaining cell viability and engraftment potential.

Erythroid differentiation and flow cytometry analysis techniques are detailed.

Abstract

Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential.

For complete details on the use and execution of this protocol, please refer to Chu et al.1

•Step-by-step guide for production and purification of DNA repair templates•Protocol for high-efficiency, precision genome editing in hematopoietic stem cells•Techniques for in vitro erythroid differentiation and flow cytometry analysis

Step-by-step guide for production and purification of DNA repair templates

Protocol for high-efficiency, precision genome editing in hematopoietic stem cells

Techniques for in vitro erythroid differentiation and flow cytometry analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential.

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