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AviTag-seq unifies nucleotide-resolution maps of CRISPR off-targets and AAV vector integrations.
Comprehensive safety assessment of gene-editing therapies requires quantifying both off-target cleavage and vector integration. However, current double-strand break (DSB)-dependent assays are fundamentally limited when evaluating nickase-based editors and are hindered by tag polarity constraints. Here, we present AviTag-seq, a platform repurposing AAV Inverted Terminal Repeats (ITRs) as universal capture tags. By exploiting the ITRs' single-stranded hairpin structure, AviTag-seq overcomes polarity issues, enabling high-sensitivity detection with a single primer pair, particularly in iPSCs. Crucially, it captures off-target events from prime and base editors that evade conventional detection. In vivo, AviTag-seq outperformed DISCOVER-Seq+ in profiling Pcsk9 off-targets in mouse liver while simultaneously mapping AAV integration sites. This dual profiling revealed that, unlike in vitro, AAV vectors in vivo preferentially integrate into active gene promoters, highlighting a specific genotoxic risk for liver-directed therapies. AviTag-seq thus offers a unified, regulatory-grade solution for evaluating diverse genome-editing modalities.
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Human germline editing: a historical perspective.
The development of the genome editing system called CRISPR-Cas9 has opened a huge debate on the possibility of modifying the human germline. But the types of changes that could and/or ought to be made have not been discussed. To cast some light on this debate, I will describe the story of the CRISPR-Cas9 system. Then, I will briefly review the projects for modification of the human species that were discussed by biologists throughout the twentieth century. Lastly, I will show that for plenty of reasons, both scientific and societal, germline modification is no longer a priority for our societies.
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Editing SOX Genes by CRISPR-Cas: Current Insights and Future Perspectives.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins (Cas) is an adaptive immune system in archaea and most bacteria. By repurposing these systems for use in eukaryote cells, a substantial revolution has arisen in the genome engineering field. In recent years, CRISPR-Cas technology was rapidly developed and different types of DNA or RNA sequence editors, gene activator or repressor, and epigenome modulators established. The versatility and feasibility of CRISPR-Cas technology has introduced this system as the most suitable tool for discovering and studying the mechanism of specific genes and also for generating appropriate cell and animal models. SOX genes play crucial roles in development processes and stemness. To elucidate the exact roles of SOX factors and their partners in tissue hemostasis and cell regeneration, generating appropriate in vitro and in vivo models is crucial. In line with these premises, CRISPR-Cas technology is a promising tool for studying different family members of SOX transcription factors. In this review, we aim to highlight the importance of CRISPR-Cas and summarize the applications of this novel, promising technology in studying and decoding the function of different members of the SOX gene family.
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Evolution in crop improvement approaches and future prospects of molecular markers to CRISPR/Cas9 system.
The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.
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The Consent Form in the Chinese CRISPR Study: In Search of Ethical Gene Editing.
This editorial provides an ethical analysis of the consent materials and other documents relating to the recent creation and birth of twin girls who had their genes edited using CRISPR-cas9 in a controversial Chinese research study. It also examines the "draft ethical principles" published by the leader of the research study. The results of the analysis further intensify serious ethical concerns about the conduct of this study.
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Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of human endometrial mesenchymal stem cells.
Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.
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Development of CRISPR/Cas9 mediated virus resistance in agriculturally important crops.
Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9) system of targeted genome editing has already revolutionized the plant science research. This is a RNA guided programmable endonuclease based system composed of 2 components, the Cas9 nuclease and an engineered guide RNA targeting any DNA sequence of the form N20-NGG for novel genome editing applications. The CRISPR/Cas9 technology of targeted genome editing has been recently applied for imparting virus resistance in plants. The robustness, wide adaptability, and easy engineering of this system has proved its potential as an antiviral tool for plants. Novel DNA free genome editing by using the preassembled Cas9/gRNA ribonucleoprotein complex for development of virus resistance in any plant species have been prospected for the future. Also, in this review we have discussed the reports of CRISPR/Cas9 mediated virus resistance strategy against geminiviruses by targeting the viral genome and transgene free strategy against RNA viruses by targeting the host plant factors. In conclusion, CRISPR/Cas9 technology will provide a more durable and broad spectrum viral resistance in agriculturally important crops which will eventually lead to public acceptance and commercialization in the near future.
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Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm.
Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.
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Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems.
Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer ∼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.
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NIH supports call for moratorium on clinical uses of germline gene editing.
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