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Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. Inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genesKIF18BandUSP28for which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack ofKIF18BorUSP28counteracts a vincristine-induced p53 response involving the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.Key pointsInhibition of the mitotic surveillance pathway (USP28-53BP1-p53) and KIF18B induces resistance to vincristineSubstantial synergistic effects observed when using the KIF18A-inhibitor BTB-1 with vincristine in eradicating GCB-subtype DLBCL cells

In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are up-regulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in up-regulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as down-regulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Retroviruses, are enveloped animal ribonucleic acid (RNA) viruses that replicate via a deoxyribonucleic acid (DNA) intermediate, which is integrated into the host genome as a provirus. Expression of viral RNA and proteins from proviral DNA utilises the transcription and translation machinery of the host. Retrovirus particles are assembled through protein–protein, protein–RNA and protein–lipid interactions, released from the cell by budding and subsequently matured by a viral protease. Interaction of the viral envelope protein with a target cell receptor triggers entry of the viral nucleoprotein core by fusion of viral and cellular membranes. After entry, the viral enzymes reverse transcriptase and integrase mediate reverse transcription of viral RNA and integration of the resulting double-stranded DNA copy of the viral genome, respectively. A provirus can be transmitted through the germ line from parents to offspring as an endogenous retrovirus. Multiple steps of retroviral replication can be targeted by host cell restriction factors in a complex interplay of virus–host interactions.

Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.

Viruses are naturally endowed with the capacity to transfer genetic material between cells. Following early skepticism, engineered viruses have been used to transfer genetic information into thousands of patients, and genetic therapies are currently attracting large investments. Despite challenges and severe adverse effects along the way, optimized technologies and improved manufacturing processes are driving gene therapy toward clinical translation. Fueled by the outbreak of AIDS in the 1980s and the accompanying focus on human immunodeficiency virus (HIV), lentiviral vectors derived from HIV have grown to become one of the most successful and widely used vector technologies. In 2022, this vector technology has been around for more than 25 years. Here, we celebrate the anniversary by portraying the vector system and its intriguing properties. We dive into the technology itself and recapitulate the use of lentiviral vectors for ex vivo gene transfer to hematopoietic stem cells and for production of CAR T-cells. Furthermore, we describe the adaptation of lentiviral vectors for in vivo gene delivery and cover the important contribution of lentiviral vectors to basic molecular research including their role as carriers of CRISPR genome editing technologies. Last, we dwell on the emerging capacity of lentiviral particles to package and transfer foreign proteins.

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

Prime editing is a new CRISPR-based genome editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with AAV viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4× fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. This novel minimized PE provides great value to AAV-based delivery applications in vivo.

Paralytic poliomyelitis is a rare disease manifestation following poliovirus (PV) infection. The disease determinants remain largely unknown. We used whole exome sequencing to uncover possible contributions of host genetics to the development of disease outcome in humans with poliomyelitis. We identified a patient with a variant in ATG7, an important regulatory gene in the macroautophagy/autophagy pathway. PV infection did not induce a prominent type I interferon response, but rather activated autophagy in neuronal-like cells, and this was essential for viral control. Importantly, virus-induced autophagy was impaired in patient fibroblasts and associated with increased viral burden and enhanced cell death following infection. Lack of ATG7 prevented control of infection in neuronal-like cells, and reconstitution of patient cells with wild-type ATG7 reestablished autophagy-mediated control of infection. Collectively, these data suggest that ATG7 defect contributes to host susceptibility to PV infection and propose autophagy as an unappreciated antiviral effector in viral infection in humans.

Prime editing (PE) is a novel CRISPR-derived genome editing technique facilitating precision editing without double-stranded DNA breaks. PE, mediated by a Cas9-reverse transcriptase fusion protein, is based on dual-functioning prime editing guide RNAs (pegRNAs), serving both as guide molecules and as templates carrying the desired edits. Due to such diverse functions, manual pegRNA design is a subject to error and not suited for large-scale setups. Here, we present pegIT, a user-friendly web tool for rapid pegRNA design for numerous user-defined edits, including large-scale setups. pegIT is freely available at https://pegit.giehmlab.dk., Graphical Abstract Graphical abstractpegIT is a freely available, user-friendly web tool that allows for easy and efficient pegRNA design for numerous, including large-scale, user-defined edits.

Background DNA transposon-based vectors are effective nonviral tools for gene therapy and genetic engineering of cells. However, promoter DNA methylation and a near-random integration profile, which can result in transgene integration into heterochromatin, renders such vectors vulnerable to transcriptional repression. Therefore, to secure persistent transgene expression it may be necessary to protect transposon-embedded transgenes with anti-transcriptional silencing elements. Results We compare four different protective strategies in CHO-K1 cells. Our findings show robust protection from silencing of transgene cassettes mediated by the ubiquitous chromatin-opening element (UCOE) derived from the HNRPA2B1-CBX3 locus. Using a bioinformatic approach, we define a shorter HNRPA2B1-CBX3 UCOE core fragment and demonstrate that this can robustly maintain transgene expression after extended passaging of CHO-K1 cells carrying DNA transposon vectors equipped with this protective feature. Conclusions Our findings contribute to the understanding of the mechanism of HNRPA2B1-CBX3 UCOE-based transgene protection and support the use of a correctly oriented core fragment of this UCOE for DNA transposon vector-based production of recombinant proteins in CHO-K1 cells.

A major clinical challenge of diffuse large B-cell lymphoma (DLBCL) is that up to 40% of patients have refractory disease or relapse after initial response to therapy as a result of drug-specific molecular resistance. The purpose of the present study was to investigate microRNA (miRNA) involvement in vincristine resistance in DLBCL, which was pursued by functional in vitro analysis in DLBCL cell lines and by outcome analysis of patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Differential miRNA expression analysis identified miR-155 as highly expressed in vincristine-sensitive DLBCL cell lines compared with resistant ones. Ectopic upregulation of miR-155 sensitized germinal-center B-cell-like (GCB)–DLBCL cell lines to vincristine, and consistently, reduction and knockout of miR-155 induced vincristine resistance, documenting that miR-155 functionally induces vincristine sensitivity. Target gene analysis identified miR-155 as inversely correlated with Wee1, supporting Wee1 as a target of miR-155 in DLBCL. Chemical inhibition of Wee1 sensitized GCB cells to vincristine, suggesting that miR-155 controls vincristine response through Wee1. Outcome analysis in clinical cohorts of DLBCL revealed that high miR-155 expression level was significantly associated with superior survival for R-CHOP-treated patients of the GCB subclass, independent of international prognostic index, challenging the commonly accepted perception of miR-155 as an oncomiR. However, miR-155 did not provide prognostic information when analyzing the entire DLBCL cohort or activated B-cell–like classified patients. In conclusion, we experimentally confirmed a direct link between high miR-155 expression and vincristine sensitivity in DLBCL and documented an improved clinical outcome of GCB-classified patients with high miR-155 expression level.

The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity., Graphical Abstract, The therapeutic effect of retinal gene therapy using CRISPR-Cas9-mediated knockout applications is dependent on efficient and safe delivery of gene-modifying tools. For the first time, Holmgaard et al. demonstrate targeted knockout of the Vegfa gene in the retina using RNPs containing SpCas9 protein complexed with chemically modified sgRNAs, suggesting that delivery of gene-disrupting agents relying on short-lived nuclease activity is a clinically relevant strategy.

Recurrent herpesvirus infections can manifest in different forms of disease, including cold sores, genital herpes, and encephalitis. There is an incomplete understanding of the genetic and immunological factors conferring susceptibility to recurrent herpes simplex virus 2 (HSV2) infection in the central nervous system (CNS). Here, we describe two adult patients with recurrent HSV2 lymphocytic Mollaret’s meningitis that each carry a rare monoallelic variant in the autophagy proteins ATG4A or LC3B2. HSV2-activated autophagy was abrogated in patient primary fibroblasts, which also exhibited significantly increased viral replication and enhanced cell death. HSV2 antigen was captured in autophagosomes of infected cells, and genetic inhibition of autophagy by disruption of autophagy genes, including ATG4A and LC3B2, led to enhanced viral replication and cell death in primary fibroblasts and a neuroblastoma cell line. Activation of autophagy by HSV2 was sensitive to ultraviolet (UV) irradiation of the virus and inhibited in the presence of acyclovir, but HSV2-induced autophagy was independent of the DNA-activated STING pathway. Reconstitution of wild-type ATG4A and LC3B2 expression using lentiviral gene delivery or electroporation of in vitro transcribed mRNA into patient cells restored virus-induced autophagy and the ability to control HSV2 replication. This study describes a previously unknown link between defective autophagy and an inborn error of immunity that can lead to increased susceptibility to HSV2 infection, suggesting an important role for autophagy in antiviral immunity in the CNS.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.

Recurrent herpesvirus infections can lead to numerous forms of diseases ranging from cold sore and genital herpes to encephalitis. Currently, there is an incomplete understanding of the genetic and immunological factors conferring susceptibility to recurrent HSV2 infections in the central nervous system (CNS). Here we describe two adult patients with recurrent HSV2 lymphocytic Mollaret’s meningitis each carrying a rare monoallelic variant in one of the autophagy proteins ATG4A and LC3B2. Importantly, HSV2-activated autophagy was abrogated in patient primary fibroblasts, which also exhibited significantly increased viral replication together with enhanced cell death. HSV2 antigen was captured in autophagosomes of infected cells, and genetic inhibition of autophagy by disruption of autophagy genes, including ATG4A and LC3B2, led to enhanced viral replication and cell death in primary fibroblasts and a neuroblastoma cell line. Activation of autophagy by HSV2 was sensitive to UV irradiation of the virus and inhibited in the presence of acyclovir, highlighting the need for a functional viral DNA genome and viral replication. However, HSV2-induced autophagy was independent of the DNA-activated STING pathway. Finally, reconstitution of wildtype ATG4A and LC3B2 expression using lentiviral gene delivery or electroporation of in vitro transcribed mRNA into patient cells restored virus-induced autophagy and the ability to control HSV2 replication. This study represents the first autophagy defect causing a primary immunodeficiency with increased susceptibility to viral infection described in humans which altogether suggests an important role for autophagy in anti-HSV2 immunity in the CNS.

A prognostic 3-miRNA classifier for early-stage mycosis fungoides has been developed recently, with miR-106b providing the strongest prognostic power. The aim of this study was to investigate the molecular function of miR-106b in mycosis fungoides disease progression. The cellular localization of miR-106b in mycosis fungoides skin biopsies was determined by in situ hybridization. The regulatory role of miR-106b was assessed by transient miR-106b inhibitor/mimic transfection of 2 mycosis fungoides derived cell lines, followed by quantitative real-time PCR (RT-qPCR), western blotting and a proliferation assay. MiR-106b was found to be expressed by dermal T-lymphocytes in mycosis fungoides skin lesions, and miR-106b expression increased with advancing mycosis fungoides stage. Transfection of miR-106b in 2 mycosis fungoides derived cell lines showed that miR-106b represses the tumour suppressors cyclin-dependent kinase inhibitor 1 (p21) and thioredoxin-interacting protein (TXNIP) and promotes mycosis fungoides tumour cell proliferation. In conclusion, these results substantiate that miR-106b has both a functional and prognostic role in progression of mycosis fungoides.

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) “conventional” tail-vein injections, (2) “primed” injections, (3) “hydrodynamic” injections, or (4) direct “intrahepatic” injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse. Keywords: lentivirus, high-pressure tail-vein injection, hydrodynamic delivery, in vivo, liver gene therapy, gene transfer, hepatocytes

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.

Adaptation of the CRISPR system has enabled scientists to probe the genome and interfere with gene function at an unprecedented scale. Adding to the use of CRISPR for generation of individual gene knockout, which is by now conventional, the CRISPR system enables high-throughput functional screening of the genome. By combining the integrative properties of lentiviral vector delivery with the disruptive nature of the CRISPR system, genome-wide CRISPR libraries provide the power to screen among thousands of genes despite the high complexity of the entire genome and identify a list of genes potentially affecting a certain phenotype. Genome-wide CRISPR screening is an advanced technology compiling numerous practical aspects and a series of molecular biology techniques. In this protocol, we describe all steps toward implementing CRISPR knockout screens in your research; we describe the core procedures and key information as well as some tricks and tips needed to successfully perform a CRISPR screen.

CRISPR, a revolutionizing technology allowing researchers to navigate in and edit the genome, is moving on the fast track toward clinical use for ex vivo correction of disease-causing mutations in stem cells. As we await the first trials utilizing ex vivo CRISPR editing, implementation of CRISPR-based gene editing as an in vivo treatment directly in patients still remains an ultimate challenge. However, quickly accumulating evidence has provided proof-of-concept for efficacious editing in vivo. Attempts to edit genes directly in animals have largely relied on classical vector systems based on virus-based delivery of gene cassettes encoding the Cas9 endonuclease and single guide RNA, the key components of the CRISPR system. However, whereas persistent gene expression has been the primary goal of gene therapy for decades, things may be different in the case of CRISPR delivery. Is short-term presence of the CRISPR components perhaps sufficient for efficacy and ideal for safety?-and are strategies needed for restricting immune recognition of the bacteria-derived editing tool? Here, while answers to these questions still blow in the wind, we review prominent examples of genome editing with focus on targeting of genes with CRISPR in liver, muscles, and eyes of the mouse.

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%-30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

Adaptation of the CRISPR system has enabled scientists to probe the genome and interfere with gene function at an unprecedented scale. Adding to the use of CRISPR for generation of individual gene knockout, which is by now conventional, the CRISPR system enables high-throughput functional screening of the genome. By combining the integrative properties of lentiviral vector delivery with the disruptive nature of the CRISPR system, genome-wide CRISPR libraries provide the power to screen among thousands of genes despite the high complexity of the entire genome and identify a list of genes potentially affecting a certain phenotype. Genome-wide CRISPR screening is an advanced technology compiling numerous practical aspects and a series of molecular biology techniques. In this protocol, we describe all steps toward implementing CRISPR knockout screens in your research; we describe the core procedures and key information as well as some tricks and tips needed to successfully perform a CRISPR screen.

CRISPR, a revolutionizing technology allowing researchers to navigate in and edit the genome, is moving on the fast track toward clinical use for ex vivo correction of disease-causing mutations in stem cells. As we await the first trials utilizing ex vivo CRISPR editing, implementation of CRISPR-based gene editing as an in vivo treatment directly in patients still remains an ultimate challenge. However, quickly accumulating evidence has provided proof-of-concept for efficacious editing in vivo. Attempts to edit genes directly in animals have largely relied on classical vector systems based on virus-based delivery of gene cassettes encoding the Cas9 endonuclease and single guide RNA, the key components of the CRISPR system. However, whereas persistent gene expression has been the primary goal of gene therapy for decades, things may be different in the case of CRISPR delivery. Is short-term presence of the CRISPR components perhaps sufficient for efficacy and ideal for safety?-and are strategies needed for restricting immune recognition of the bacteria-derived editing tool? Here, while answers to these questions still blow in the wind, we review prominent examples of genome editing with focus on targeting of genes with CRISPR in liver, muscles, and eyes of the mouse.