Protein-polymer Core-shell Structured Nanocarriers For Cytosolic Delivery Of Cas9/sgRNA

CRISPR/Cas9 has become the most important gene editing tool in recent years,which enabling researchers to manipulate specific DNA sequences in organisms and cell types with ease,efficiency,and cost-effectiveness.Direct cytosolic delivery of Cas9/sg RNA can avoid uncontrolled integrational mutagenesis and unexpected off-target adverse effects which mainly caused by gene delivery strategies.These advantages will improve the safety of CRISPR/Cas9 technology and show broad prospects in clinical applications.However,currently available Cas9/g RNA delivery vectors are associated with complicated synthetic routes,low gene-editing efficiency,and cytotoxicity from insufficient degradation.More importantly,none of these vectors have been used in hard-to-transfect cell types(e.g.,primary T cells).In this study,we tackled above mentioned issues with a new type of nanomaterials to achieve direct cytosolic delivery of Cas9/sg RNA and gene editing in living cells.Proteins are regarded as natural “carriers”.We first synthesized a novel protein-polymer core-shell nanostructure by in situ radical polymerization.Designable surface modification enables proteins to act as delivery vectors for negatively charged biomacromolecules.Cationic protein-polymer nanocarriers show good loading capacity for negatively charged biomacromolecules such as plasmid DNA through non-covalent interactions.The mechanistic study reveals that modification with tertiary amine groups facilitates the endosomal escape of complexes,and the subsequent hydrolysis of biodegradable ester linkages between the cationic unit and the opposite end of the polymer under physiological conditions triggers the cytosolic release of biomacromolecules from nanocarriers.The nanocarrier B4 with 2-(dimethylamino)ethyl methacrylate(DMAEMA)as the cationic monomer,and N,N′-methylenebisacrylamide(BIS)as the crosslinker is highly noticeable,affording ～80% transfection efficiency and stable protein expression for over four days.A cell model expressing single copy of the EGFP-PEST reporter gene was established for high-throughput screening of Cas9/sg RNA delivery vectors.After successful cytosolic delivery of Cas9/sg RNA targeting EGFP,the EGFP gene in cell models was disrupted,the EGFP-PEST fusion proteins degraded rapidly,and the fluorescence signals decreased,indicating the occurrence of gene editing.Gene editing can be detected in 2–4 days by quantitative or qualitative measurements by flow cytometry with good visualization.A Cas9/sg RNA delivery system based on protein-polymer core-shell nanocarriers was constructed using the cell evaluation model.First,the delivery efficiency of B4 was evaluated.Results showed that B4 carried Cas9/sg RNA into cells with internalization efficiency at 100%,and gene-editing efficiency below 5%,which suggesting that Cas9/sg RNA complexes were not effectively released.To further optimize the design scheme and parameters of the DMAEMA material systems,we designed nanocarriers with similar compositions to B4.The nanocarrier B5 without crosslinker affords the highest gene-editing efficiency of ～40% in cell models despite a lower Cas9/sg RNA internalization level than B4.Then,the release mechanism of Cas9/sg RNA from B5 and the structural effect of B5 on the gene editing performance were elucidated by simulating the intracellular physiological environment in vitro.It is suggested that the instability of B5 itself and the binding competition from cytosolic anionic species trigger the release of Cas9/sg RNA.Moreover,enhanced Cas9/sg RNA stability is achieved for B5 as entrapment in its star-like structure enables Cas9/sg RNA protection against enzymatic hydrolysis,which is superior to B4 with a crosslinked capsule-like structure.Finally,B5 based Cas9/sg RNA delivery was performed in T cells.Cell activation plays an important role in Cas9/sg RNA internalization.The cellular uptake of Cas9/sg RNA for the “Reactivated-3D” cells was about 6 times that of non-activated cells,resulting in ～14% gene disruption.Aiming at the therapeutic target of T-cell malignancies(CD7)and the immune checkpoint inhibitor(PD1),B5 mediated Cas9/sg RNA delivery achieved effective gene editing in T cells.Herein,we developed a Cas9/sg RNA delivery nanocarrier system based on protein-polymer structure through rational design and screening.This work provides a new class of self-assembled platforms for Cas9/sg RNA delivery,and further details the parameters for the future vector design and cytosolic release kinetics control.The methodology we proposed is hoped to provide a new alternative gene-knockout approach for next-generation CAR-T immunotherapies.