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VERVE-101: a promising CRISPR-based gene editing therapy that reduces LDL-C and PCSK9 levels in HeFH patients.
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Consumer acceptance of new plant-breeding technologies: An application to the use of gene editing in fresh table grapes.
This study estimates consumers' willingness to pay for specific product (quality) and process (agronomic) attributes of table grapes, including taste, texture, external appearance, and the expected number of chemical applications, and for the breeding technology used to develop the plant. Considering varietal traits, on average our survey respondents were willing to pay the highest price premiums for specific offers of improvements in table grape taste and texture, followed by external appearance and expected number of chemical applications. Considering breeding methods, on average our respondents were willing to pay a small premium for table grapes developed using conventional breeding rather than gene editing (e.g., CRISPR). Results from a latent class model identify four different groups of consumers with distinct preferences for grape quality attributes and breeding technologies. The group of consumers most likely to reject gene editing considers both genetic engineering and gene editing to be breeding technologies that produce foods that are morally unacceptable and not safe to eat.
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CRISPR/Cas tool designs for multiplex genome editing and its applications in developing biotic and abiotic stress-resistant crop plants.
Crop plants are prone to several yield-reducing biotic and abiotic stresses. The crop yield reductions due to these stresses need addressing to maintain an adequate balance between the increasing world population and food production to avoid food scarcities in the future. It is impossible to increase the area under food crops proportionately to meet the rising food demand. In such an adverse scenario overcoming the biotic and abiotic stresses through biotechnological interventions may serve as a boon to help meet the globe's food requirements. Under the current genomic era, the wide availability of genomic resources and genome editing technologies such as Transcription Activator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and Clustered-Regularly Interspaced Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas) has widened the scope of overcoming these stresses for several food crops. These techniques have made gene editing more manageable and accessible with changes at the embryo level by adding or deleting DNA sequences of the target gene(s) from the genome. The CRISPR construct consists of a single guide RNA having complementarity with the nucleotide fragments of the target gene sequence, accompanied by a protospacer adjacent motif. The target sequence in the organism's genome is then cleaved by the Cas9 endonuclease for obtaining a desired trait of interest. The current review describes the components, mechanisms, and types of CRISPR/Cas techniques and how this technology has helped to functionally characterize genes associated with various biotic and abiotic stresses in a target organism. This review also summarizes the application of CRISPR/Cas technology targeting these stresses in crops through knocking down/out of associated genes.
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ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila.
Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system ("ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.
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Microinjection for precision genome editing in Caenorhabditis elegans.
In Caenorhabditis elegans, targeted genome editing techniques are now routinely used to generate germline edits. The remarkable ease of C. elegans germline editing is attributed to the syncytial nature of the pachytene ovary which is easily accessed by microinjection. This protocol describes the step-by-step details and troubleshooting tips for the entire CRISPR-Cas genome editing procedure, including gRNA design and microinjection of ribonucleoprotein complexes, followed by screening and genotyping in C. elegans, to help accessing this powerful genetic animal system. For complete details on the use and execution of this protocol, please refer to Ghanta and Mello (2020).
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Broad Institute wins bitter battle over CRISPR patents.
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Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.
An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.
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Protocol for transformation-free genome editing in plants using RNA virus vectors for CRISPR-Cas delivery.
Plant virus vectors have emerged as promising tools for CRISPR-Cas reagent delivery. Here, we present a protocol for DNA-free plant genome editing using an engineered RNA virus vector for the transient delivery of CRISPR-Cas components. We describe steps for viral vector construction, viral vector recovery through agroinoculation of Nicotiana benthamiana, mechanical inoculation of target plant hosts, analysis of somatic mutagenesis frequency, and regeneration of mutant plants. The method achieves high editing efficiency and eliminates the need for stable plant transformation. For complete details on the use and execution of this protocol, please refer to Liu et al.1.
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Smart Strategies for Precise Delivery of CRISPR/Cas9 in Genome Editing.
The emergence of CRISPR/Cas technology has enabled scientists to precisely edit genomic DNA sequences. This approach can be used to modulate gene expression for the treatment of genetic disorders and incurable diseases such as cancer. This potent genome-editing tool is based on a single guide RNA (sgRNA) strand that recognizes the targeted DNA, plus a Cas nuclease protein for binding and processing the target. CRISPR/Cas has great potential for editing many genes in different types of cells and organisms both in vitro and in vivo. Despite these remarkable advances, the risk of off-target effects has hindered the translation of CRISPR/Cas technology into clinical applications. To overcome this hurdle, researchers have devised gene regulatory systems that can be controlled in a spatiotemporal manner, by designing special sgRNA, Cas, and CRISPR/Cas delivery vehicles that are responsive to different stimuli, such as temperature, light, magnetic fields, ultrasound (US), pH, redox, and enzymatic activity. These systems can even respond to dual or multiple stimuli simultaneously, thereby providing superior spatial and temporal control over CRISPR/Cas gene editing. Herein, we summarize the latest advances on smart sgRNA, Cas, and CRISPR/Cas nanocarriers, categorized according to their stimulus type (physical, chemical, or biological).
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A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment Engineering in Acremonium chrysogenum.
Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy. Acremonium chrysogenum remains the only microorganism capable of industrial-scale CPC production to date. However, the lack of efficient multiplex genome-editing tools has limited studies on its gene function, high-yield mechanisms as well as metabolic engineering. To overcome this limitation, a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system was developed, driven by endogenous tRNA promoters, enabling one-step multilocus knockout, large-fragment DNA deletion, and gene overexpression in A. chrysogenum. Given that many strains lack visible phenotypes associated with specific genes, we introduced a visually distinguishable red phenotype by expressing the heterologous protein mCherry under a strong promoter. In the wild-type strain, 20 endogenous tRNA promoters were evaluated and compared to the heterologous Aspergillus nidulans PgpdA and Aspergillus fumigatus U6 promoters. The endogenous tRNAVal promoter showed the highest knockout efficiency (95.5%). The tRNA-gRNA array-based CRISPR/Cas9 system enabled double- and triple-site knockouts without donor DNA in industrial strain, with efficiencies of 50.0-83.3% and 14.3%, respectively. This is the first demonstration of simultaneous triple-site knockout in A. chrysogenum, especially in industrial strain. Using this system, we successfully deleted a 50.7-kb DNA fragment containing the sorbicillinoids biosynthetic gene cluster with nearly 100% efficiency and achieved overexpression of the key gene pcbAB involved in CPC biosynthesis in high-yield strain, increasing CPC titer from 5.59 g/L to 6.92 g/L with an improvement of 23.8%. Overall, this tRNA-gRNA array-based CRISPR/Cas9 multiplex gene-editing system provides an efficient and versatile platform for functional genomics and industrial strain engineering in A. chrysogenum.
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