Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells

Figures

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  Fig. 1. Comparison of different biomolecular CRISPR/Cas9 formats. In the process of delivering plasmid DNA, it is essential that the plasmid reaches the nucleus. Here, the transcription mechanism activates, converting the gene into gRNA and Cas9 mRNA. Following this, in the cytoplasm, Cas9 mRNA undergoes translation to produce Cas9 protein. Subsequently, both gRNA and Cas9 protein are transported back into the nucleus, where the CRISPR mechanism can enact its effect on the targeted genomic DNA. Alternatively, for Cas9 mRNA delivery, the cargo must be released into the cytosol. This allows for the translation of mRNA into Cas9 protein to occur. Notably, the Ribonucleoprotein (RNP) delivery method offers instantaneous results compared to other strategies. By bypassing the translation and transcription processes, gene editing can commence immediately upon delivery. (This figure was created by the authors using Adobe Photoshop software)

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  Fig. 2. Schematic of strategies for delivering Cas9 RNP complex to HSCs. a. TRIAMF Filtroporation Device. Composed of a silicone washer, stainless-steel mesh, polycarbonate filter membrane, and PTFE washer. The syringe reservoir contains the RNP and HSC mixture, which is propelled through the membrane using nitrogen pressure into a culture plate. b. Polymer Nanoparticles (PNPs) for RNP Delivery. The figure illustrates the use of PNPs to deliver RNP complexes. The depicted polymeric structures have been proven safe and efficient for delivering CRISPR-Cas9 system components, with controllable speed, timing, and location of delivery. c. Microfluidic-Based Nano-Blade Chip. This figure showcases the Nano-Blade chip, constructed from silicon instead of the traditional PDMS. The chip’s unique design includes an asymmetrical microchannel with a silicon nanoblade on one side of the deformation zone. This structure applies contact pressure to CD34 + HSPCs, disrupting their membrane for efficient macro-molecule or plasmid delivery. d. An Illustration of the Electroporation Process. This image demonstrates the electro-physical technique of electroporation, a swift and non-viral method used for the…