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The era of Cas12 and Cas13 CRISPR-based disease diagnosis.
Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems, since their discovery, have found growing applications in cell imaging, transcription modulation, therapeutics and diagnostics. Discovery of Cas12 and Cas13 have brought a new dimension to the field of disease diagnosis. These endonucleases have been extensively used for diagnosis of viral diseases in humans and animals and to a lesser extent in plants. The exigency of SARS-CoV-2 pandemic has highlighted the potential of CRISPR-Cas systems and sparked the development of innovative point-of-care diagnostic technologies. Rapid adaptation of CRISPR-chemistry combined with sensitive read-outs for emerging pathogens make them ideal candidates for detection and management of diseases in future. CRISPR-based approaches have been recruited for the challenging task of cancer detection and prognosis. It stands to reason that the field of CRISPR-Cas-based diagnosis is likely to expand with Cas12 and Cas13 playing a pivotal role. Here we focus exclusively on Cas12- and Cas13-based molecular diagnosis in humans, animals and plants including the detection of SARS-coronavirus. The CRISPR-based diagnosis of plant and animal diseases have not found adequate mention in previous reviews. We discuss various advancements, the potential shortfalls and challenges in the widespread adaptation of this technology for disease diagnosis.
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Development of a comprehensive set of tools for genome engineering in a cold- and thermo-tolerant Kluyveromyces marxianus yeast strain.
Kluyveromyces marxianus, a non-conventional thermotolerant yeast, is potentially useful for production of ethanol and other products. This species has a strong tendency to randomly integrate transforming DNA fragments, making necessary the development of more precise methods for gene targeting. In this study, we first demonstrated that K. marxianus NBRC1777 is cold-tolerant, and then established a highly efficient and precise technique for gene editing by introducing genes encoding deaminase-mediated targeted point mutagenesis (Target-AID) and clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas9). We used Target-AID to introduce targeted point mutations that disrupted Nej1 or Dnl4, genes that are involved in non-homologous end-joining (NHEJ). Both of the resulting mutant strains showed enhanced proportions of homology-mediated integration compared to the wild-type parent. In combination with target cleavage by CRISPR-Cas9, markerless integration was performed using short (~50 bp) flanking homologous sequences. Together, these tools render this species fully tractable for gene manipulation, permitting targeted genetic changes in the cold- and thermo-tolerant yeast K. marxianus.
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Genome editing in Sub-Saharan Africa: a game-changing strategy for climate change mitigation and sustainable agriculture.
Sub-Saharan Africa's agricultural sector faces a multifaceted challenge due to climate change consisting of high temperatures, changing precipitation trends, alongside intensified pest and disease outbreaks. Conventional plant breeding methods have historically contributed to yield gains in Africa, and the intensifying demand for food security outpaces these improvements due to a confluence of factors, including rising urbanization, improved living standards, and population growth. To address escalating food demands amidst urbanization, rising living standards, and population growth, a paradigm shift toward more sustainable and innovative crop improvement strategies is imperative. Genome editing technologies offer a promising avenue for achieving sustained yield increases while bolstering resilience against escalating biotic and abiotic stresses associated with climate change. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) is unique due to its ubiquity, efficacy, alongside precision, making it a pivotal tool for Sub-Saharan African crop improvement. This review highlights the challenges and explores the prospect of gene editing to secure the region's future foods.
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CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot 'Kurodagosun' resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot 'Deep purple', resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.
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Cell-derived extracellular vesicles for CRISPR/Cas9 delivery: engineering strategies for cargo packaging and loading.
Genome editing technology has emerged as a potential therapeutic tool for treating incurable diseases. In particular, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems and the design of single-guide RNAs (sgRNAs) have revolutionized genome editing applications. Unfortunately, compared with the rapid development of gene-editing tools, the progress in the development of delivery technologies is lagging behind and thus limiting the clinical application of genome editing. To overcome these limitations, researchers have investigated various delivery systems, including viral and non-viral vectors for delivering CRISPR/Cas and sgRNA complexes. As natural endogenous nanocarriers, extracellular vesicles (EVs) present advantages of biocompatibility, low immunogenicity, stability, and high permeability, making them one of the most promising drug delivery vehicles. This review provides an overview of the fundamental mechanisms of EVs from the aspects of biogenesis, trafficking, cargo delivery, and function as nanotherapeutic agents. We also summarize the latest trends in EV-based CRISPR/Cas delivery systems and discuss the prospects for future development. In particular, we put our emphasis on the state-of-the-art engineering strategies to realize efficient cargo packaging and loading. Altogether, EVs hold promise in bridging genome editing in the laboratory and clinical applications of gene therapies by providing a safe, effective, and targeted delivery vehicle.
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Gene Editing for Cystic Fibrosis: Advances and Prospects of CRISPR-Cas9 Therapy.
Cystic fibrosis (CF) is an inherited, autosomal recessive disorder that is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CFTR maintains the balance between water and salts by transporting chloride ions along various epithelial surfaces. CFTR impairment affects the function of several organs, including the lungs. Newborn screening, prenatal diagnosis, and pharmacological interventions have altered the prevalence and incidence of cystic fibrosis. Although CFTR modulators are a promising treatment option, their ability to target and correct only one mutation at a time restricts their therapeutic potential. The development of genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas(CRISPR-Cas9) has the potential to correct genetic mutations, including those associated with CF, thereby offering a permanent treatment by fixing the root cause of CF. This article summarizes cystic fibrosis development, prognosis, and diagnosis, as well as possibilities for correcting various types of CFTR gene mutations. The review focuses on the potential of gene editing technologies to repair CFTR mutations and their applications in the advancement of CF treatment.
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Optimization of genome engineering approaches with the CRISPR/Cas9 system.
Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.
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Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks.
Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.
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Gene therapy for huntington's disease: advances, challenges, and future perspectives.
An abnormal amplification of the CAG trinucleotide repeat in the huntingtin (HTT) gene causes Huntington’s disease (HD), a devastating neurological illness. The striatum and cortex are primarily affected by the gradual neuronal dysfunction and loss caused by the mutant huntingtin (mHTT) protein. Although there have been notable improvements in symptomatic management, curative treatments remain unavailable. A promising treatment option that offers specifically designed interventions to decrease or eradicate mutant HTT expression is gene therapy. RNA-targeted treatments (antisense oligonucleotides, RNA interference), DNA editing methods (Clustered Regularly Interspaced Short Palindromic Repeats/caspase 9, zinc finger nucleases, Transcription Activator-like Effector Nuclease), and advanced delivery systems (viral and non-viral vectors, lipid nanoparticles, exosomes) are just a few of the many approaches that are presently being researched. AMT-130 and tominersen clinical trials provide crucial information about the viability, security, and effectiveness of gene therapy for HD. It is hoped that developments in genome editing and delivery methods would make gene therapy a viable treatment for HD. However, challenges remain, including immunological responses, blood-brain barrier penetration, and off-target consequences. This review delves into the latest developments in HD gene therapy, highlighting new approaches, obstacles, and potential future paths to a permanent cure.
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Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9.
Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.
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