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Back to Basics: Application of the Principles of Bioethics to Heritable Genome Interventions.
Prior to their announcement of the birth of gene-edited twins in China, Dr. He Jiankui and colleagues published a set of draft ethical principles for discussing the legal, social, and ethical aspects of heritable genome interventions. Within this document, He and colleagues made it clear that their goal with these principles was to "clarify for the public the clinical future of early-in-life genetic surgeries" or heritable genome editing. In light of He's widely criticized gene editing experiments it is of interest to place these draft principles in the larger ethical debate surrounding heritable genome editing. Here we examine the principles proposed by He and colleagues through the lens of Beauchamp and Childress' Principles of Biomedical Ethics. We also analyze the stated goal that the "clinical future" of heritable genome editing was clarified by He and colleagues' proposed principles. Finally, we highlight what might be done to help prevent individual actors from pushing forward ahead of broad societal consensus on heritable genome editing.
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Development of an in vivo cleavable donor plasmid for targeted transgene integration by CRISPR-Cas9 and CRISPR-Cas12a.
The CRISPR-Cas system is widely used for genome editing of cultured cells and organisms. The discovery of a new single RNA-guided endonuclease, CRISPR-Cas12a, in addition to the conventional CRISPR-Cas9 has broadened the number of editable target sites on the genome. Here, we developed an in vivo cleavable donor plasmid for precise targeted knock-in of external DNA by both Cas9 and Cas12a. This plasmid, named pCriMGET_9-12a (plasmid of synthetic CRISPR-coded RNA target sequence-equipped donor plasmid-mediated gene targeting via Cas9 and Cas12a), comprises the protospacer-adjacent motif sequences of Cas9 and Cas12a at the side of an off-target free synthetic CRISPR-coded RNA target sequence and a multiple cloning site for donor cassette insertion. pCriMGET_9-12a generates a linearized donor cassette in vivo by both CRISPR-Cas9 and CRISPR-Cas12a, which resulted in increased knock-in efficiency in culture cells. This method also achieved > 25% targeted knock-in of long external DNA (> 4 kb) in mice by both CRISPR-Cas9 and CRISPR-Cas12a. The pCriMGET_9-12a system expands the genomic target space for transgene knock-in and provides a versatile, low-cost, and high-performance CRISPR genome editing tool.
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Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing.
Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.
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Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells.
The CRISPR-Cas9 system is a genomic editing tool widely used in basic research and under investigation for potential applications in gene therapies for human diseases. To accomplish genomic editing, the system requires the expression of a prokaryotic DNA endonuclease enzyme, Cas9, in host cells. Previous studies have mainly focused on the specificity of Cas9 on the host genome, and thus it is unclear whether this bacterium-derived enzyme affects the protein homeostasis of host cells. Here we applied multi-omic analyses, including transcriptome, proteome, phosphoproteome, Cas9-associated protein interactome, protein synthesis, and histone epigenetic modification, to investigate the cellular response of human cells upon the expression of Cas9. We demonstrate that Cas9 has minimal impact on host cells. Our assessment of intracellular effects of Cas9 paves a path for its broad applications in biological studies and potential clinical translations.
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High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli.
Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including fimA, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in E. coli.
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Breeding by Design for Functional Rice with Genome Editing Technologies.
The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.
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Characterization and Engineering of a Novel Miniature Eubacterium siraeum CRISPR-Cas12f System.
Cas12f nucleases are one of the most compact genome editors, exhibiting promising potential for in vivo therapeutic applications. However, the availability of active Cas12f genome editors remains relatively limited in the field. Here, we report the characterization and engineering of a novel miniature Cas12f endonuclease from Eubacterium siraeum (EsCas12f1, 433 amino acids). We elucidate the specific Protospacer Adjacent Motifs preference and the detailed biochemical properties for DNA targeting and cleavage. By employing rational design strategies, we systematically optimize the guide RNA of EsCas12f1, converting the initially ineffective CRISPR-EsCas12f1 system into an efficient bacterial genome editor. Furthermore, we demonstrate the capacity of EsCas12f1 for in vitro nucleic-acid diagnostics. In summary, our results enrich the miniature CRISPR-Cas toolbox and pave the way for the application of EsCas12f1 for both genome editing and in vitro diagnostics.
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Genome-editing technologies: the gap between application and policy.
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Expanded precision genome-editing toolbox for human disease modeling in zebrafish.
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ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology.
Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease.
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