Key Takeaways & Executive Findings
- •• CRISPR-Cas RNP complexes provide a non-viral, efficient delivery method for gene editing in hematopoietic stem cells, offering rapid activity and reduced off-target effects. • Novel delivery techniques including microfluidic-based methods, filtroporation, nanoparticles, and cell-penetrating peptides are advancing the clinical application of CRISPR-Cas RNP for hematologic disorder therapies. • The review highlights the potential of RNP delivery to overcome hurdles in HSC gene editing, paving the way for safer and more effective genetic treatments. • Optimizing delivery strategies is crucial for translating CRISPR-Cas RNP technology into clinical practice for hematologic diseases.
Abstract
Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.
1. Introduction
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus, a 1664-nucleotide sequence, was first discovered in E. coli in 1987 [1]. Although various studies have explored this gene locus and similar loci found in different bacteria and archaea species [2–5], the primary function of this locus remained unknown until 2007. The exploration of genes adjacent to the CRISPR locus [6] and the identification of foreign viral DNA within the CRISPR locus spacers prompted the hypothesis that this locus functions as an adaptive immune system in bacteria and archaea, defending against viral invasions [7, 8]. This hypothesis was confirmed by the Barrangou Lab in 2007 [9].
Due to its ability to precisely target and cut specific DNA sequences, the CRISPR-Cas system was recognized as a genome editing tool in 2012 [10]. This system performs much better than tools previously used, such as meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Its design and implementation are much simpler, and it has broader applications [11]. The CRISPR-Cas9 system, Type II from S. pyogenes, is the most common variant among this system’s two types and six subtypes [12]. It creates specific double-strand breaks (DSBs) and facilitates targeted genome editing [13].
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Zahra Molaei, Zahra Jabbarpour, Azadeh Omidkhoda, Naser Ahmadbeigi (2026). Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells. Stem Cell Research & Therapy. https://doi.org/10.1186/s13287-024-03848-4
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Frequently Asked Questions
What is the CRISPR-Cas system?
The CRISPR-Cas system is a genome editing tool derived from a bacterial adaptive immune system. It uses a guide RNA to direct the Cas9 nuclease to specific DNA sequences, creating double-strand breaks for targeted gene modification.
Why is the delivery of CRISPR-Cas to hematopoietic stem cells challenging?
Hematopoietic stem cells are sensitive and difficult to transfect efficiently. Viral vectors can have safety concerns, while non-viral methods often suffer from low delivery efficiency or toxicity. The RNP complex offers a safer alternative but still requires effective delivery methods.
What are the advantages of using RNP complexes for gene editing?
RNP complexes provide enhanced gene editing capabilities, are devoid of viral vectors, have rapid activity, and minimize off-target effects compared to other delivery formats.
What novel delivery methods are being explored for RNP complexes?
Novel methods include microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides, which aim to improve delivery efficiency and safety.
What is the significance of this review?
This review comprehensively summarizes current and emerging non-viral delivery methods for CRISPR-Cas RNP to HSCs, highlighting their potential for treating hematologic disorders and guiding future research.
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