Abstract
The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.
Highlights
Due to its simplicity and adaptability, the CRISPR/Cas[9] system has rapidly become one of the most popular approaches for genome engineering and is accelerating the study of many biological systems
We report on the development of a hybrid system, combining traceable lentiviral single guide RNA (sgRNA) delivery with transient delivery of Cas[9] for CRISPR/Cas[9] genome editing in primary human hematopoietic stem and progenitor cells (HSPCs)
By delivering Cas[9] as mRNA and using a modified sgRNA backbone, c hRNA28,22, we show that this approach is highly flexible and scalable, allowing for efficient editing with low toxicity and preserved stem and progenitor cell potential
Summary
Due to its simplicity and adaptability, the CRISPR/Cas[9] system has rapidly become one of the most popular approaches for genome engineering and is accelerating the study of many biological systems. It has been reported that modified sgRNAs enhance genome editing in C D34+ HSPCs, demonstrating an advantage over unmodified sgRNAs in human cells when co-delivered with Cas[9] mRNA or delivered as RNP complex due to their increased stability[17]. These approaches do not allow tracing of the edited cells. 7. (d) Maintenance of EGFP expression following transduction and EGFP sorting of C D34+ HSPCs. Data from two independent transductions (TD 1 and TD 2) (e) Overview of experimental outline for gene editing in. CD34+ HSPCs using lentiviral delivery of sgRNA targeting CD45 and electroporation of Cas[9] mRNA or protein
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