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1. Reversible RNA acylation for control of CRISPR–Cas9 gene editing

   https://doi.org/10.1039/C9SC03639C  

   Habibian, Maryam ; McKinlay, Colin ; Blake, Timothy R. ; Kietrys, Anna M. ; Waymouth, Robert M. ; Wender, Paul A. ; Kool, Eric T. ( January 2020 , Chemical Science)

   null (Ed.)

   We report the development of post-transcriptional chemical methods that enable control over CRISPR–Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N 3 ) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated (“cloaked”) sgRNA was completely inactive in DNA cleavage with Cas9 in vitro , and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR–Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function. 

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2. In Vivo Editing of Macrophages through Systemic Delivery of CRISPR‐Cas9‐Ribonucleoprotein‐Nanoparticle Nanoassemblies

   https://doi.org/10.1002/adtp.201900041  

   Lee, Yi‐Wei ; Mout, Rubul ; Luther, David C. ; Liu, Yuanchang ; Castellanos‐García, Laura ; Burnside, Amy S. ; Ray, Moumita ; Tonga, Gulen Yeşilbag ; Hardie, Joseph ; Nagaraj, Harini ; et al ( August 2019 , Advanced Therapeutics)

   Macrophages are key effectors of host defense and metabolism, making them promising targets for transient genetic therapy. Gene editing through the delivery of Cas9‐ribonucleoprotein (RNP) provides multiple advantages over gene delivery–based strategies for introducing CRISPR machinery to the cell. There are, however, significant physiological, cellular, and intracellular barriers to the effective delivery of the Cas9 protein and guide RNA (sgRNA) that have to date, restricted in vivo Cas9 protein–based approaches to local/topical delivery applications. Described herein is a new nanoassembled platform featuring coengineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9‐sgRNA delivery and concomitant CRISPR editing through systemic tail‐vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.

    

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3. mRNA Delivery Using Bioreducible Lipidoid Nanoparticles Facilitates Neural Differentiation of Human Mesenchymal Stem Cells

   https://doi.org/10.1002/adhm.202000938  

   Zhao, Xuewei ; Glass, Zachary ; Chen, Jinjin ; Yang, Liu ; Kaplan, David L. ; Xu, Qiaobing ( August 2020 , Advanced Healthcare Materials)

   Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and tissue engineering and delivering biological molecules into MSCs has been used to control stem cell behavior. However, the efficient delivery of large biomolecules such as DNA, RNA, and proteins into MSCs using nonviral delivery strategies remains an ongoing challenge. Herein, nanoparticles composed of cationic bioreducible lipid‐like materials (lipidoids) are developed to intracellularly deliver mRNA into human mesenchymal stem cells (hMSCs). The delivery efficacy to hMSCs is improved by adding three excipients including cholesterol, 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐polyethylene glycol (DSPE‐PEG) during lipidoid nanoparticle formulation. Using an optimized lipidoid formulation, Cas9 mRNA and single guide RNA (sgRNA) targeting neuron restrictive silencing factor (NRSF) are delivered to hMSCs, leading to successful neural‐like differentiation as demonstrated by the expression of synaptophysin (SYP), brain‐derived neurotrophic factor (BDNF), neuron‐specific enolase (NSE), and neuron‐specific growth‐associated protein (SCG10). Overall, a synthetic lipid formulation that can efficiently deliver mRNA to hMSCs is identified, leading to CRISPR‐based gene knockdown to facilitate hMSCs transdifferentiation into neural‐like lineage.

    

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4. Efficient Multi‐Allelic Genome Editing of Primary Cell Cultures via CRISPR‐Cas9 Ribonucleoprotein Nucleofection

   https://doi.org/10.1002/cpsc.126  

   Hoellerbauer, Pia ; Kufeld, Megan ; Paddison, Patrick J. ( August 2020 , Current Protocols in Stem Cell Biology)

   CRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR‐Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2′‐O‐methyl‐3′phosphorothioate‐modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion‐deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs). This article provides examples of the nucleofection buffer and programs that are optimal for patient‐derived glioblastoma (GBM) stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs), but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem‐like cells. © 2020 Wiley Periodicals LLC

   Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion‐deletion (indel) mutation and genomic deletion generation in primary cell cultures

    

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5. A Versatile Nonviral Delivery System for Multiplex Gene‐Editing in the Liver

   https://doi.org/10.1002/adma.202003537  

   Gong, Jing ; Wang, Hong‐Xia ; Lao, Yeh‐Hsing ; Hu, Hanze ; Vatan, Naazanene ; Guo, Jonathan ; Ho, Tzu‐Chieh ; Huang, Dantong ; Li, Mingqiang ; Shao, Dan ; et al ( October 2020 , Advanced Materials)

   Recent advances in CRISPR present attractive genome‐editing toolsets for therapeutic strategies at the genetic level. Here, a liposome‐coated mesoporous silica nanoparticle (lipoMSN) is reported as an effective CRISPR delivery system for multiplex gene‐editing in the liver. The MSN provides efficient loading of Cas9 plasmid as well as Cas9 protein/guide RNA ribonucleoprotein complex (RNP), while liposome‐coating offers improved serum stability and enhanced cell uptake. Hypothesizing that loss‐of‐function mutation in the lipid‐metabolism‐related genespcsk9,apoc3, andangptl3would improve cardiovascular health by lowering blood cholesterol and triglycerides, the lipoMSN is used to deliver a combination of RNPs targeting these genes. When targeting a single gene, the lipoMSN achieved a 54% gene‐editing efficiency, besting the state‐of‐art Lipofectamine CRISPRMax. For multiplexing, lipoMSN maintained significant gene‐editing at each gene target despite reduced dosage of target‐specific RNP. By delivering combinations of targeting RNPs in the same nanoparticle, synergistic effects on lipid metabolism are observed in vitro and vivo. These effects, such as a 50% decrease in serum cholesterol after 4 weeks of post‐treatment with lipoMSN carrying bothpcsk9andangptl3‐targeted RNPs, could not be reached with a single gene‐editing approach. Taken together, this lipoMSN represents a versatile platform for the development of efficient, combinatorial gene‐editing therapeutics.

    

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