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Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.
In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.
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CRISPR-Cas9 Gene-Edited Therapy Shows Sustained Treatment Response.
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Science and Bioethics of CRISPR-Cas9 Gene Editing: An Analysis Towards Separating Facts and Fiction.
Since its emergence in 2012, the genome editing technique known as CRISPR-Cas9 and its scientific use have rapidly expanded globally within a very short period of time. The technique consists of using an RNA guide molecule to bind to complementary DNA sequences, which simultaneously recruits the endonuclease Cas9 to introduce double-stranded breaks in the target DNA. The resulting double-stranded break is then repaired, allowing modification or removal of specific DNA bases. The technique has gained momentum in the laboratory because it is cheap, quick, and easy to use. Moreover, it is also being applied in vivo to generate more complex animal model systems. Such use of genome editing has proven to be highly effective and warrants a potential therapy for both genetic and non-genetic diseases. Although genome editing has the potential to be a transformative therapy for patients it is still in its infancy. Consequently, the legal and ethical frameworks are yet to be fully discussed and will be an increasingly important topic as the technology moves towards more contentious issues such as modification of the germline. Here, we review a number of scientific and ethical issues which may potentially influence the development of both the technology and its use in the clinical setting.
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Therapeutic Genome Editing With CRISPR/Cas9 in a Humanized Mouse Model Ameliorates α1-antitrypsin Deficiency Phenotype.
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
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Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9.
Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.
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[CRISPR/Cas9 System and Its Advances in Gene Therapy of Hematologic Diseases--Review].
In recent years, with the development of gene editing technology, the site-specific genome can be modified. The curability of genetic disease may be achieved by the use of gene editing techniques. As the simplicity, high specificity and economical efficiency, much attention has been paid to the CRISPR/Cas9 system, which was been widely used in research of molecular biology and other fields of life science. In this review, the mechanism for CR1SPR/Cas9 system and the progress of gene therapy, such as for hemophilia, betathalassaemia and chronic myeloid leukemia were summarized briefly.
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Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.
Metabolic engineering frequently requires both gene knockouts and gene integration. CRISPR-Cas9 has been extensively used to create double-stranded DNA breaks that result in indel mutations; however, such mutations can revert or create toxic product. Gene integration can also be accomplished by CRISPR-Cas9 introduced double-stranded DNA breaks and a donor DNA cassette. Here we describe our protocol for combining an efficient gene knockout created by introducing DNA cuts with two guide RNAs with a gene to be integrated at the knockout site. Including guide RNA target sites flanking the homology regions around the gene to be integrated enables both homology-directed repair and homology-mediated end joining, resulting in few deletions and a significant proportion of correctly knocked out and integrated genes.
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An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing.
Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/ybx1) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.
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Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment.
Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.
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Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos.
Microinjection is predominantly used to produce genetically modified fish. However, there are certain difficulties involved in some fish species. In this study, we tested an alternative method to produce genetically modified zebrafish by delivering DNA and other materials into embryos by electroporation. We optimized the electroporation conditions of a square wave electroporation system that work efficiently for the introduction of plasmid DNA, recombinant Cas9 nuclease and synthetic dual guide RNAs. Transgenic expression was observed in a wide range of tissues, which is comparable with those obtained by microinjection. We further determined that efficient gene delivery can be achieved during the cleavage stage. This study describes detailed electroporation parameters for gene delivery with high efficiency and low toxicity, providing a novel method to generate transgenic lines and genome editing.
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