An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling ß-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle ß-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous ß-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons.

Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function.This article has an associated First Person interview with the first author of the paper.

Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions.

Recent advances in fluorescence microscopy allow three-dimensional analysis of HIV-1 preintegration complexes in the nuclei of infected cells. To extend this investigation to gammaretroviruses, we engineered a fluorescent Moloney murine leukemia virus (MLV) system consisting of MLV-integrase fused to enhanced green fluorescent protein (MLV-IN-EGFP). A comparative analysis of lentiviral (HIV-1) and gammaretroviral (MLV) fluorescent complexes in the nuclei of infected cells revealed their different spatial distributions. This research tool has the potential to achieve new insight into the nuclear biology of these retroviruses.

Reduction of HIV-1 infectivity through endoplasmic reticulum-associated degradation-mediated Env depletion.

During the HIV-1 replicative cycle, the gp160 envelope is processed in the secretory pathway to mature into the gp41 and gp120 subunits. Misfolded proteins located within the endoplasmic reticulum (ER) are proteasomally degraded through the ER-associated degradation (ERAD) pathway, a quality control system operating in this compartment. Here, we exploited the ERAD pathway to induce the degradation of gp160 during viral production, thus leading to the release of gp120-depleted viral particles.

CD4 and BST-2/tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules.

CD4 and BST-2/Tetherin are cellular membrane proteins targeted to degradation by the HIV-1 protein Vpu. In both cases proteasomal degradation following recruitment into the ERAD pathway has been described. CD4 is a type I transmembrane glycoprotein, with four extracellular immunoglobulin-like domains containing three intrachain disulfide bridges. BST-2/Tetherin is an atypical type II transmembrane glycoprotein with an N-terminal transmembrane domain and a C-terminal glycophosphatidylinositol anchor, which dimerizes through three interchain bridges. We investigated spontaneous and Vpu-induced retro-translocation of CD4 and BST-2/Tetherin using our novel biotinylation technique in living cells to determine ER-to-cytosol retro-translocation of proteins. We found that CD4 retro-translocates with oxidized intrachain disulfide bridges, and only upon proteasomal inhibition does it accumulate in the cytosol as already reduced and deglycosylated molecules. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric oxidized complex and then becomes deglycosylated and reduced to monomers. These results raise questions on the required features of the putative retro-translocon, suggesting alternative retro-translocation mechanisms for membrane proteins in which complete cysteine reduction and unfolding are not always strictly required before ER to cytosol dislocation.

EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.

Eukaryotic-driven directed evolution of Cas9 nucleases.

BACKGROUND: Further advancement of genome editing highly depends on the development of tools with higher compatibility with eukaryotes. A multitude of described Cas9s have great potential but require optimization for genome editing purposes. Among these, the Cas9 from Campylobacter jejuni, CjCas9, has a favorable small size, facilitating delivery in mammalian cells. Nonetheless, its full exploitation is limited by its poor editing activity. RESULTS: Here, we develop a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA is validated with CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 is up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. CONCLUSIONS: We report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.

Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data.

The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement.

CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells.

hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).

Author Correction: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice.

Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. This construct, called ClopHensor, consists of a H+- and Cl--sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) ß chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9.

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology.

A highly specific SpCas9 variant is identified by in vivo screening in yeast.

Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.

Hit and go CAS9 delivered through a lentiviral based self-limiting circuit.

In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.