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Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.
The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.
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Genome scale screening identification of SaCas9/gRNAs for targeting HIV-1 provirus and suppression of HIV-1 infection.
The CRISPR/Cas9 gene-editing approach has been widely used in anti-HIV-1 gene therapy research. However, the major challenges facing the therapeutic application of CRISPR/Cas9 are the precise genome cleavage efficacy and efficient delivery of Cas9/gRNA specifically to the HIV-infected cells. Recently, a small size Cas9 from Staphylococcus aureus (SaCas9) has shown promise in genome editing in eukaryotic cells, suggesting a potential usage in blocking HIV-1 infection by targeting the HIV-1 genome. Here, we designed 43 guide RNAs (gRNAs) against the HIV-1 genome, thereby identifying 8 gRNAs that efficiently and specifically disrupt the target DNA by SaCas9. In addition, we found the selected gRNAs induce SaCas9 to disrupt the latent HIV-1 provirus and suppress HIV-1 proviral reactivation in latently infected Jurkat C11 cells. We further confirmed that the dual or triple gRNAs in an all-in-one lentiviral vector could reduce viral production in TZM-bl cells as well as in Jurkat T cells. Moreover, we did not detect any off-target cleavages in the predicted sites, suggesting that through all-in-one lentiviral vector-mediated HIV-1 genome editing, the selected SaCas9/gRNAs can provide an alternative and flexible strategy for anti-HIV gene therapy.
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Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.
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Keynote Symposium.
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CRISPR/Cas9 and the future of clinical research.
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Thermozymes: Adaptive strategies and tools for their biotechnological applications.
In today's scenario of global climate change, there is a colossal demand for sustainable industrial processes and enzymes from thermophiles. Plausibly, thermozymes are an important toolkit, as they are known to be polyextremophilic in nature. Small genome size and diverse molecular conformational modifications have been implicated in devising adaptive strategies. Besides, the utilization of chemical technology and gene editing attributions according to mechanical necessities are the additional key factor for efficacious bioprocess development. Microbial thermozymes have been extensively used in waste management, biofuel, food, paper, detergent, medicinal and pharmaceutical industries. To understand the strength of enzymes at higher temperatures different models utilize X-ray structures of thermostable proteins, machine learning calculations, neural networks, but unified adaptive measures are yet to be totally comprehended. The present review provides a recent updates on thermozymes and various interdisciplinary applications including the aspects of thermophiles bioengineering utilizing synthetic biology and gene editing tools.
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Cloning and validating systems for high throughput molecular recording.
Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.
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CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos.
CRISPR-Cas9 genome editing has transformed biology by enabling site-specific genome modifications to be simply engineered. Here, we describe two CRISPR-Cas9 approaches to introduce MS2 stem-loop sequences into endogenous gene loci in Drosophila. This can facilitate live imaging of nascent transcription in Drosophila. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).
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Prime Editing Exhibits Limited Genome-Wide Off-Target Effects in Cellular and Embryonic Gene Editing.
Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use.
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Efficient genome engineering in Agrobacterium tumefaciens C58 using recombineering assisted by CRISPR/Cas9.
Recombineering, a technique derived from phage-encoded homologous recombination, has emerged as a vital approach for bacterial genome engineering. Agrobacterium tumefaciens is extensively utilized to transfer DNA into the host plant genomes. To facilitate the transformation of various plant species, particularly those of considerable economic value, genetic modifications of Agrobacterium strains are essential. Our previous studies established an Agrobacterium-specific phage-encoded homologous recombination system for Agrobacterium species. Yet, recent investigations have indicated that there is a substantial variability in the recombination efficiency of these recombineering systems for gene editing across different genome loci in A. tumefaciens. In this work, we present the development of an efficient genome engineering tool for A. tumefaciens by integrating recombineering with CRISPR/Cas9 technology. Initially, we found that lengthening the homology arms significantly enhanced genome editing efficiency. Nevertheless, at certain genomic sites, even when the length of the homology arms was increased, the editing efficiency remained suboptimal. Subsequently, combination of the Agrobacterium-specific recombineering system with the CRISPR/Cas9 system markedly enhanced the genome engineering efficiency. This study offers an enhanced and efficient genome engineering tool for A. tumefaciens, which could potentially be applied to other species within the Agrobacterium genus.
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