Site Specific Knock-In Genome Editing in Human HSCs Using Baboon Envelope gp Pseudotypedviral Derived “Nanoblades” Loaded with Cas9/sgRNA Combined with Donor Encoding AAV-6

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. Here, we have designed ?Nanoblades?, a new technology that will deliver a genomic cleaving agent into cells. These are genetically modified Murine Leukemia Virus (MLV) or HIV derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9. These VLPs are thus loaded with Cas9 protein together with the guide RNAs. Thus, nanoblades are devoid of any viral-derived genetic material. Highly efficient gene editing was obtained in cell lines, IPS cells and primary mouse and human cells (Mangeot et al. Nature Communication, 2019). However, their delivery into target cells can be technically challenging when working with primary immune cells. Now we showed that nanoblades were remarkably efficient for entry into human T, B and hematopoietic stem cells thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. We were able to induce efficient, transient and very rapidlygenome-editing in human induced pluripotent stem cells reaching up to 70% in the empty spiracles homeobox 1 (EMX1) and muscular dystrophy (MD) gene locus. A brief nanoblade incubation of primary human T and B cells resulted in 40% and 20% editing of the Wiskott-Aldrich syndrome (WAS) gene locus, while hematopoietic stem cells treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target. Moreover, for the HIV- and MLV-derived nanoblades no cell toxicity and low to undetectable off-target effects were demonstrated.Finally, we also treated hHSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus. Currently, we are evaluating these gene modified HSCs for their long-term reconstitution of NOD/SCIDgC-/- mice.Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy.Fil: Gutierrez, Alejandra. Inserm; FranciaFil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina. Inserm; FranciaFil: Mangeot, Philippe E.. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornellie. Inserm; FranciaFil: Fusil, Floriane. Inserm; FranciaFil: Froment, Gisèle. Inserm; FranciaFil: Martin, Francisco. Inserm; FranciaFil: Bellabdelah, Karim. Universidad de Granada; EspañaFil: Ricci, Emiliano P.. Inserm; FranciaFil: Ayuso, Eduard. Universite de Nantes; FranciaFil: Cosset, François loic. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaAmerican Society of Cell and Gene Therapy 22nd Annual MettingWashingtonEstados UnidosAmerican Society of Cell and Gene Therap

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication.

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Nanoblades allow high-level genome editing in murine and human organoids

Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called “organoids” is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the “nanoblade (NB)” technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%–50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression

IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity

A Versatile Strategy to Reduce UGA-Selenocysteine Recoding Efficiency of the Ribosome Using CRISPR-Cas9-Viral-Like-Particles Targeting Selenocysteine-tRNA[Ser]Sec Gene

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism

A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium

The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression

Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor

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