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Found: 26 165
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Comparison of Prime Editing and CRISPR-Cas Mediated HDR in Correcting CFTR-F508del Mutation in Patient iPSC-Derived Airway Basal Cells.
Gene editing therapy represents a promising strategy to permanently cure cystic fibrosis (CF). This paper presents the results of correcting the most common CF mutation, F508del, in the CFTR gene in airway basal cells (BCs) obtained from patients, using both classical CRISPR-Cas9-mediated homology-directed repair (HDR) and prime editing techniques. Our findings demonstrate that both methods achieve mutation correction with efficiencies reaching up to 18.9% of alleles in successfully transfected cells. However, the CRISPR-Cas9 approach results in insertions and deletions (indels) in approximately 15% of alleles at the editing site, whereas prime editing induced only minimal indel events. Functional analysis evaluating the effectiveness of gene editing revealed that prime editing of the F508del mutation in the CFTR gene-using the pegRNA4 combined with the PE2-NG variant-restores CFTR channel conductance, comparable to triple therapy with CFTR modulators (ivacaftor/tezacaftor/elexacaftor). While both prime editing and CRISPR-HDR resulted in successful correction of the CFTR-F508del mutation in patient BCs, prime editing exhibited a low frequency of unintended modifications both within and outside the editing locus, making this approach a promising candidate for the development of gene therapy strategies for CF.
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Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript.
The CRISPR-Cas system is a well-established RNA-guided DNA editing technique widely used to modify genomic DNA sequences. I used the CRISPR-Cas9 system to change the second and third nucleotides of the triplet TCT of human TNSFSF9 in HepG2 cells to TAG to create an amber stop codon. The TCT triplet is the codon for Ser at the 172nd position of TNSFSF9. The two substituted nucleotides, AG, were confirmed by DNA sequencing of the PCR product followed by PCR amplification of the genomic TNFSF9 gene. Interestingly, sequencing of the cDNA of transcripts of the edited TNFSF9 gene revealed that the TAG had been re-edited to the wild type triplet TCT, and 1 or 2 bases just before the triplet had been deleted. These observations indicate that CRISPR-Cas9-mediated editing of bases in target genomic DNA can be followed by spontaneous re-editing (correcting) of the bases during transcription.
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Targeted candidate gene screens using CRISPR/Cas9 technology.
In the postgenomic era, the ability to quickly, efficiently, and inexpensively assign function to the zebrafish proteome is critical. Clustered regularly interspaced short palindromic repeats (CRISPRs) have revolutionized the ability to perform reverse genetics because of its simplicity and broad applicability. The CRISPR system is composed of an engineered, gene-specific single guide RNA (sgRNA) and a Cas9 enzyme that causes double-stranded breaks in DNA at the targeted site. This simple, two-part system, when injected into one-cell stage zebrafish embryos, efficiently mutates target loci at a frequency such that injected embryos phenocopy known mutant phenotypes. This property allows for CRISPR-based F0 screening in zebrafish, which provides a means to screen through a large number of candidate genes for their role in a phenotype of interest. While there are important considerations for any successful genetic screen, CRISPR screening has significant benefits over conventional methods and can be accomplished in any lab with modest molecular biology experience.
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Research Note: Injection of adenoviral CRISPR/Cas9 system targeting melanophilin gene into different sites of embryos induced regional feather color changes in posthatch quail.
Poultry species is an important animal model in both avian research and the poultry industry. To advance our understanding of genetic factors and benefit both fields, a gene of interest can be genetically edited, and consequential phenotypic changes can be investigated. Injection of adenovirus containing the CRISPR/Cas9 system into avian blastoderm induced genome editing in blastodermal cells randomly, including primordial germ cells, which results in generation of whole-body knockout in the offspring of the virus-injected quail. However, to observe phenotypic and functional changes in whole-body, homozygous knockout of genes using this genome editing technology requires at least 2 generations of breeding of chimeric, and heterozygotes birds. In the current study, we developed a strategy to investigate the gene function in 1-generation by inducing regional genome editing around the injection sites with CRISPR/Cas9 adenovirus. The adenoviral CRISPR/Cas9 vector targeting the melanophilin (Mlph) gene, regulating feather pigmentation, was injected into 2 different regions of embryos, the cervical flexure of quail embryos at HH stage 13 to 15 and the tip of the upper limb bud of embryos at HH stage 22 to 24, to induce genome editing in those regions. Indel mutations in the target loci of the Mlph gene were detected by extracting genomic DNA from the embryonic tissues, and consequential phenotypes, feather color changes, were analyzed at 1 mo after hatch. Injection of the adenovirus into the cervical flexure and the tip of the upper limb bud of embryos resulted in 8 to 21% efficiency of indel mutation in the embryonic cells of the injected regions. In the posthatch quail, gray feathers were shown on their upper back and primary wing feathers, corresponding to the injection sites at embryos. Successful validation of this strategy for inducing genome editing in parts of tissues within 1-generation will accelerate studies on genetic functions with advantages of less time and cost, facilitating avian research and providing foundations for future application for the poultry industry.
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CRISPR gene editing in human embryos wreaks chromosomal mayhem.
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Efficient production of gene-edited onion (Allium cepa) plants using biolistic delivery of cas9 RNPs and transient expression constructs.
Delivery of Cas9/sgRNA RNPs and DNA vectors designed to transiently express morphogenic regulatory genes or an antibiotic resistance gene enabled efficient recovery of gene edited onion lines. We developed a protocol for efficiently producing gene edited onion plants that does not depend on stable transformation. The process makes use of transient gene expression to enrich for gene editing among plants regenerated from immature embryos bombarded with ribonucleoprotein (RNP) complexes composed of CRISPR-associated protein 9 (Cas9) and single guide RNAs (sgRNAs). We used the Allium cepa Downy Mildew Resistant 6 (AcDMR6) gene as the target gene for our studies and produced a total of 47 onion plants with edited AcDMR6 alleles, including 13 homozygous plants, 12 biallelic plants, 7 heterozygous plants, and 15 chimeric plants. The most effective strategy for producing gene edited onion plants involved co-delivering plasmids encoding a hygromycin-resistance protein and plant developmental regulator genes along with the Cas9/sgRNA RNPs followed by transient Hygromycin selection for 48 h. Using this approach, up to 12% of the regenerated onion plants carried edited alleles of AcDMR6. By comparison, no editing was observed among the 146 plants regenerated from explants transfected with the Cas9/sgRNA RNPs alone. The strategy we describe here for using transient gene expression to enrich for gene editing in onion could potentially be extended to other crop species as well.
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Challenges and opportunities in gene editing of B cells.
B cells have long been an underutilized target in immune cell engineering, despite a number of unique attributes that could address longstanding challenges in medicine. Notably, B cells evolved to secrete large quantities of antibodies for prolonged periods, making them suitable platforms for long-term protein delivery. Recent advances in gene editing technologies, such as CRISPR-Cas, have improved the precision and efficiency of engineering and expanded potential applications of engineered B cells. While most work on B cell editing has focused on ex vivo modification, a body of recent work has also advanced the possibility of in vivo editing applications. In this review, we will discuss both past and current approaches to B cell engineering, and its promising applications in immunology research and therapeutic gene editing.
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CRISPR-based gene therapy dampens pain in mice.
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[Stochastic Packaging of Cas Proteins into Exosomes].
CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.
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Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection.
The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/μL Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1-5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.
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