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Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.
The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.
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What history tells us XL. The success story of the expression 'genome editing'.
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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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Rational Design of Small Molecules to Enhance Genome Editing Efficiency by Selectively Targeting Distinct Functional States of CRISPR-Cas12a.
CRISPR-Cas12a, a type-V CRISPR-Cas endonuclease, is an effective genome editing platform. To improve the gene editing efficiency of Cas12a, we rationally designed small molecule enhancers through a combined computational approach. First, we used extensive molecular dynamics (MD) simulations to explore the conformational landscape of Cas12a from Acidaminococcus (AsCas12a), revealing distinct conformational states that could be targeted by small molecules to modulate its genome editing function. We then identified 57 compounds that showed different binding behavior and stabilizing effects on these distinct conformational states using molecular docking. After experimental testing 6 of these 57 compounds, compound 1, quinazoline-2,4(1H,3H)-dione, was found particularly promising in enhancing the AsCas12a-mediated genome editing efficiency in human cells. Compound 1 was shown to act like a molecular "glue" at the interface between AsCas12a and crRNA near the 5'-handle region, thus specifically stabilizing the enzyme-crRNA complex. These results provide a new paradigm for future design of small molecules to modulate the genome editing of the CRISPR-Cas systems.
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CRISPR/Cas bioimaging: From whole body biodistribution to single-cell dynamics.
This review explores the transformative role of CRISPR/Cas systems in optical bioimaging, emphasizing how advancements in nanoparticle (NP) technologies are revolutionizing the visualization of gene-editing processes both in vitro and in vivo. Optical imaging techniques, such as near-infrared (NIR) and fluorescence imaging, have greatly benefited from the integration of nanoformulated contrast agents, improving resolution, sensitivity, and specificity. CRISPR/Cas systems, originally developed just for gene editing, are now being coupled with these imaging modalities to enable real-time monitoring and quantitative measurements of metabolites, vitamins, proteins, nucleic acids and other entities in specific areas of the body, as well as tracking of CRISPR/Cas delivery, editing efficiency, and potential off-target effects. The development of CRISPR/Cas-loaded NPs allows for enhanced imaging and precise monitoring across multiple scales with multiplexed and multicolor imaging in complex settings, including potential in vivo diagnostics. CRISPR/Cas therapeutics as well as diagnostics are hindered by the lack of efficient and targeted delivery tools. Biomimetic NPs have emerged as promising tools for improving biocompatibility, enhancing targeting capabilities, and overcoming biological barriers, facilitating more efficient delivery and bioimaging of CRISPR/Cas systems in vivo. As the design of these NPs and delivery mechanisms improves, alongside advancements in endolysosomal escape, CRISPR/Cas-based bioimaging will continue to advance, offering unprecedented possibilities in precision medicine and theranostic applications.
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Consumer acceptance of food crops developed by genome editing.
One of the major problems regarding consumer acceptance of genetically modified organisms (GMOs) is the possibility that their transgenes could have adverse effects on the environment and/or human health. Genome editing, represented by the CRISPR/Cas9 system, can efficiently achieve transgene-free gene modifications and is anticipated to generate a wide spectrum of plants. However, the public attitude against GMOs suggests that people will initially be unlikely to accept these plants. We herein explored the bottlenecks of consumer acceptance of transgene-free food crops developed by genome editing and made some recommendations. People should not pursue a zero-risk bias regarding such crops. Developers are encouraged to produce cultivars with a trait that would satisfy consumer needs. Moreover, they should carefully investigate off-target mutations in resultant plants and initially refrain from agricultural use of multiplex genome editing for better risk-benefit communication. The government must consider their regulatory status and establish appropriate regulations if necessary. The government also should foster communication between the public and developers. If people are informed of the benefits of genome editing-mediated plant breeding and trust in the relevant regulations, and if careful risk-benefit communication and sincere considerations for the right to know approach are guaranteed, then such transgene-free crops could gradually be integrated into society.
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CRISPR based genome editing and removal of human viruses.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins 9 (Cas9), a gene-editing technology, has been extensively applied as a tool for genetic engineering in basic research. Efficient genome engineering has been performed in viruses, human cells, bacteria, fungi, plants and animals, etc. Currently, it has been employed to edit human viruses for studying viral molecular biology, pathogenesis and oncogenesis, and facilitate the development of antiviral agents and vaccine. The virus is ubiquitous worldwide and elicits global health problems, many human diseases are associated with virus infections. Although traditional drugs can be used to treat or prevent productive viral infections, their efficacy is limited because of toxicity, side effects and other problems. Additionally, no current drugs are approved to be indicated for latent infections. Therefore, the next highlight is to develop antiviral approaches to against both productive and latent infections. Fortunately, CRISPR has been successfully applied in the removal of human viruses ex vivo and/or in vivo, and has the potential to be used to manufacture antiviral agents for clinical application. CRISPR/Cas9 is promising in applications, even though some technical challenges, safety concerns, ethic concerns need to be improved. In this article, the discovery and application of genome editing and removal of human viruses based on CRISPR are explored. Additionally, we evaluate the prospects and limitations of this novel antiviral strategies.
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CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide RNAs.
OBJECTIVE: To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei. RESULTS: Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%. CONCLUSIONS: Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.
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Spacer-Complementary Single-Stranded DNA Oligonucleotides Can Serve as Target-Specific Inhibitors in CRISPR/Cas9 Systems.
The continuous expression of the CRISPR/Cas system in organisms can lead to various potential issues. Some anti-CRISPR strategies have been developed to achieve precise control over CRISPR/Cas, yet these strategies are predominantly protein-based, with the most commonly used anti-CRISPR (Acr) proteins lacking sufficient target specificity. However, in this study, we designed a single-stranded DNA (ssDNA) inhibitor that was complementary to the spacer region on the guide RNA, operating at the nucleic acid level. We demonstrated that this method effectively inhibits the cleavage activity of Cas9-sgRNA ribonucleoprotein (RNP) in a target-specific manner through in vitro cleavage experiments. Furthermore, we explored the binding position and effective length of this inhibitory ssDNA, finding that its inhibitory effect was significantly reduced when the length of continuous complementarity with the 5' end of the spacer was less than 7nt. The truncated ssDNA also showed potential in reducing off-target effects. Moreover, we applied nucleic acid inhibitors to embryos via microinjection, and gene editing activity was significantly reduced, as evidenced by a decrease in the mosaicism rate of mouse embryos undergoing normal gene editing from (84.4 ± 4.4) % to 0%. Our study introduces a convenient and target-specific nucleic acid inhibitor capable of achieving precise regulation of gene editing.
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Developing Striga resistance in sorghum by modulating host cues through CRISPR/Cas9 gene editing.
High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E0 lines in the E1 generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.
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