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      Selective ORgan Targeting (SORT) nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing

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          Abstract

          CRISPR/Cas gene editing and messenger RNA (mRNA)-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed Selective ORgan Targeting (SORT) wherein multiple classes of lipid nanoparticles (LNPs) are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen-, and liver-targeted SORT LNPs were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells, and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, and is envisioned to aid development of protein replacement and gene correction therapeutics in targeted tissues.

          Reporting summary.

          Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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          Most cited references53

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Multiplex genome engineering using CRISPR/Cas systems.

            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              RNA-guided human genome engineering via Cas9.

              Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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                Author and article information

                Journal
                101283273
                34218
                Nat Nanotechnol
                Nat Nanotechnol
                Nature nanotechnology
                1748-3387
                1748-3395
                5 March 2020
                06 April 2020
                April 2020
                14 December 2020
                : 15
                : 4
                : 313-320
                Affiliations
                [1 ]The University of Texas Southwestern Medical Center, Department of Biochemistry, Simmons Comprehensive Cancer Center, Dallas, United States.
                Author notes

                Author contributions

                Q.C., T.W., and D.J.S. conceived and designed the experiments and wrote the manuscript. Q.C., T.W., L.F., L.T.J., and S.A.D. performed experiments. All authors discussed the results and commented on the manuscript. D.J.S. directed the research.

                [†]

                These authors contributed equally to this work.

                Article
                NIHMS1564244
                10.1038/s41565-020-0669-6
                7735425
                32251383
                1d434ffd-59ce-49c9-bc7c-e54e1dc02eab

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                Nanotechnology
                Nanotechnology

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