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Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.
The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.
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Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.
Mutations in microRNA-96 (MIR96) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for the MIR96 mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A/+ mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human MIR96 mutations. Because mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by MIR96 mutations.
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Design and Generation of Drosophila Single Guide RNA Expression Constructs.
The recent advances in CRISPR-based genome engineering have enabled a plethora of new experiments to study a wide range of biological questions. The major attraction of this system over previous methods is its high efficiency and simplicity of use. For example, whereas previous genome engineering technologies required the generation of new proteins to target each new locus, CRISPR requires only the expression of a different single guide RNA (sgRNA). This sgRNA binds to the Cas9 endonuclease protein and directs the generation of a double-strand break to a highly specific genomic site determined by the sgRNA sequence. In addition, the relative simplicity of the Drosophila genome is a particular advantage, as possible sgRNA off-target sites can easily be avoided. Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications.
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Cloning and functional verification of endogenous U6 promoters for developing an efficient CRISPR/Cas9-mediated genome editing system in kenaf (Hibiscus cannabinus L.).
BACKGROUND: The U6 promoter is a critical component of the CRISPR/Cas9 system, as it drives the transcription of single-guide RNAs (sgRNAs) to enable precise genome editing. Endogenous promoters typically exhibit higher transcriptional activity than their exogenous counterparts, which can significantly enhance editing efficiency. However, the endogenous U6 promoter in kenaf (Hibiscus cannabinus L.), an important fiber crop, has not yet been characterized. METHODS: Using the Arabidopsis U6-26 (AtU6-26) promoter as a reference, we performed a homologous sequence search and identified two candidate U6 promoters in kenaf, designated HcU6-1 and HcU6-14. Promoter fragments were amplified from the kenaf cultivar 'Fuhong 952' and cloned into a β-glucuronidase (GUS) reporter vector. Histochemical GUS staining assays revealed that both HcU6 promoters were transcriptionally active, with HcU6-14 showing significantly stronger expression levels compared to HcU6-1. RESULTS: To further evaluate the utility of these promoters for genome editing, we constructed CRISPR/Cas9 vectors targeting the kenaf acetolactate synthase (ALS) gene, driven by either HcU6-14P or the exogenous cotton GbU6-9P promoter. Agrobacterium rhizogenes K599-mediated transformation was used to induce hairy roots, and mutation analysis of the ALS gene was performed via Sanger sequencing. Notably, targeted mutations in the ALS gene were detected in hairy roots transformed with the HcU6-14P-driven CRISPR/Cas9 vector, whereas no mutations were observed in roots transformed with the exogenous GbU6-9P promoter. These results demonstrate that the endogenous HcU6-14 promoter confers superior genome editing efficiency compared to the heterologous promoter, which facilitates the development of improved varieties with enhanced agronomic traits.
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Research into overcoming drug resistance in lung cancer treatment using CRISPR-Cas9 technology: a narrative review.
BACKGROUND AND OBJECTIVE: Lung cancer remains a leading cause of cancer-related mortality globally, with drug resistance posing a significant challenge to effective treatment. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) technology offers a novel and precise gene-editing technology for targeting and negating drug resistance mechanisms in lung cancer. This review summarizes the research progress in the use of CRISPR-Cas9 technology for investigating and managing drug resistance in lung cancer treatment. METHODS: A literature search was conducted using the Web of Science and PubMed databases, with the following keywords: [CRISPR-Cas9], [lung cancer], [drug resistance], [gene editing], and [gene therapy]. The search was limited to articles published in English from 2002 to September 2023. From the search results, studies that utilized CRISPR-Cas9 technology in the context of lung cancer drug resistance were selected for further analysis and summarize. KEY CONTENT AND FINDINGS: CRISPR-Cas9 technology enables precise DNA-sequence editing, allowing for the targeted addition, deletion, or modification of genes. It has been applied to investigate drug resistance in lung cancer by focusing on key genes such as epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), tumor protein 53 (TP53), and B-cell lymphoma/leukemia-2 (BCL2), among others. The technology has shown potential in inhibiting tumor growth, repairing mutations, and enhancing the sensitivity of cancer cells to chemotherapy. Additionally, CRISPR-Cas9 has been used to identify novel key genes and molecular mechanisms contributing to drug resistance, offering new avenues for therapeutic intervention. The review also highlights the use of CRISPR-Cas9 in targeting immune escape mechanisms and the development of strategies to improve drug sensitivity. CONCLUSIONS: The CRISPR-Cas9 technology holds great promise for advancing lung cancer treatment, particularly in addressing drug resistance. The ability to precisely target and edit genes involved in resistance pathways offers a powerful tool for developing more effective and personalized therapies. While challenges remain in terms of delivery, safety, and ethical considerations, ongoing research and technological refinements are expected to further enhance the role of CRISPR-Cas9 in improving patient outcomes in lung cancer treatment.
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Using Clustered Regularly Interspaced Short Palindromic Repeats gene editing to induce permanent expression of fetal hemoglobin in β-thalassemia and sickle cell disease: A comparative meta-analysis.
β-hemoglobinopathies like sickle cell disease (SCD) and β-thalassemia are characterized by differing mutations in the hemoglobin subunit beta gene (HBB). These disorders vary in phenotypic presentation and severity, with more severe manifestations leading to transfusion dependence along with associated complications such as infection and iron overload. β-hemoglobinopathies symptoms rapidly worsen after birth as the levels of fetal hemoglobin (HbF) begin to decline. To reverse this decline, current treatment plans typically involve the use of pharmacological agents such as hydroxyurea to raise expression levels of HbF. However, these treatments only result in transient effects and must be consistently administered. Gene editing technologies such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated protein) offer the opportunity to create novel treatments which can raise HbF expression with potential permanent effects. Two gene targets, B-cell lymphoma/leukemia 11A gene (BCL11A) and the promoter regions of gamma globin genes (HBG1/2), have been identified to significantly increase HbF protein expression. In order to differentiate the effectiveness of BCL11A and HBG1/2 editing, a meta-analysis was performed by first identifying 119 studies for inclusion based on the search terms terms "β-Thalassemia," "beta-thal" "sickle cell disease," "SCD," and "CRISPR." Following application of exclusion and inclusion criteria, we performed analysis on 8 peer-reviewed published studies from 2018 to 2021 were included in the study. Forest plots were generated using R (version 4.1.2). Primary comparative analysis shows HBG1/2 had a significantly (p < 0.01)greater impact on induction of HbF expression compared to BCL11A. This analysis leads us to conclude that HBG1/2 merits further investigation as a possible gene editing target for treatment of SCD and β-thalassemia.
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Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents.
Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
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i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.
We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.
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Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.
Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.
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Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.
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