Abstract
The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.
Highlights
IntroductionThe protein has proved itself to be highly specific and simple to use but has been utilized in combination with a plethora of different fusion proteins, where novel functions have been added to Cas9 [1]
Cas9 protein, clustered regularly interspaced short palindromic repeat (CRISPR) RNA, tracr RNA, single-guide RNA, and oligonucleotides were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA)
The cells incubated with PF14-AB showed a large increase in mean fluorescent intensity and percentage positive cells over the naked AB, Supplementary Materials Figure S2A,B, the signal increase correlated with the size of the attached fluorescent tag
Summary
The protein has proved itself to be highly specific and simple to use but has been utilized in combination with a plethora of different fusion proteins, where novel functions have been added to Cas9 [1]. These functions include, but are not limited to, gene regulation and de novo gene DNA repair pathways [2,3,4,5]. Cas has enabled a new beginning for gene editing, and while Cas may not be the final protein chosen for the mainstream treatment of a genetic disorder, the protein chosen is likely to be a CRISPR derivative. Several clinical studies are being performed or have been completed utilizing
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