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A visual monitoring DNA-free multi-gene editing system excised via LoxP::FRT/FLP in poplar.
The CRISPR-Cas9 system has emerged as a potent molecular scalpel for precise genome editing, and profoundly revolutionized plant genetics and breeding, facilitating the development of innovative and improved plant varieties. Typically, the CRISPR-Cas9 gene-editing construct is delivered into target organisms via Agrobacterium tumefaciens-mediated transformation or biolistic methods. However, the incorporation of the CRISPR-Cas9 machinery increases the risk of off-target effects, causing unintended genomic alterations. Additionally, the introduction of exogenous DNA sequences, such as antibiotic resistance marker, raises public concerns regarding the biosafety and regulatory oversight of genetically modified organisms (GMOs), potentially hindering regulatory approval and commercialization. Here, we have engineered an integrated system comprising RUBY, LoxP::FRT/FLP and CRISPR/Cas9-sgRNA cassettes within a single construct, allowing visible color monitoring throughout process including genetic transform, positive transgenic and edited events screening, as well as exogenous DNA excision events, we refer to it as 'a visual monitoring DNA-free multi-gene editing system (VMDFGE)'. This system was introduced into poplar through Agrobacterium tumefaciens-mediated transformation, yielding transgenic poplars with a 75.0% visual screening rate, a 45.8% targeted mutation rate and a 54.5% excision rate for the entire integration system. This approach eliminates the concerns associated with off-target effects and GMO regulatory challenges. It offers significant potential for improvement of poplar, other woody plants and crop species while removing the foreign DNA.
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CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans.
Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.
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CRISPR-Cas9 Gene Editing to Generate Isoform-Specific LAMP-2A Knockout in Human Cancer Cells.
Chaperone-mediated autophagy (CMA) is a highly specific lysosomal-dependent protein degradation pathway. A critical molecular component of CMA is the lysosome-associated membrane protein (LAMP) type 2A, which is required for substrate uptake by the lysosome. Defects in the CMA pathway have been associated with various human pathologies, including malignancies, increasing the overall interest in methods to monitor this selective autophagy process. Yet isogenic LAMP-2A knockout cancer cell models are still lacking. This is likely to depend on challenges related to that human LAMP-2 gene undergoes alternative splicing of its pre-mRNA, generating three isoform variants, LAMP-2A, LAMP-2B, and LAMP-2C. However, without assessment of the impact of LAMP-2A loss of function specifically in human cells, the involvement of CMA in human pathologies, including carcinogenesis remains speculative. Here, we describe the generation of isoform-specific CRISPR-Cas9 genomic editing of LAMP-2A in human cancer cells, without affecting the other two isoforms, allowing for experimental evaluation of LAMP-2A, thus CMA in human cancer models.
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Regeneration of Transgenic Nicotiana benthamiana Raised from the Genome-Edited Protoplast.
Plant genome editing is an emerging technique that has revolutionized plant genome engineering which helps to edit the plant genome precisely for the development of traits in many crops. Specifically, with clustered regularly interspaced short palindromic sequence (CRISPR)-CRISPR-associated protein (Cas) system, a progressive improvement in genome editing has been achieved with protoplast. Though protoplast isolation, transfection, and regeneration are available for many plants, regeneration of protoplast for many plants remains major challenge. In this methodology chapter, we outlined the construction of sgRNA for genome editing, transfection, and regeneration of transgenic N. benthamiana from the genome-edited protoplast and assay for gene targeting.
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65 YEARS OF THE DOUBLE HELIX: The advancements of gene editing and potential application to hereditary cancer.
Hereditary cancer predisposition syndromes are associated with germline mutations that lead to increased vulnerability for an individual to develop cancers. Such germline mutations in tumour suppressor genes, oncogenes and genes encoding for proteins essential in DNA repair pathways and cell cycle control can cause overall chromosomal instability in the genome and increase risk in developing cancers. Gene correction of these germline mutations to restore normal protein functions is anticipated as a new therapeutic option. This can be achieved through disruption of gain-of-function pathogenic mutation, restoration of loss-of-function mutation, addition of a transgene essential for cell function and single nucleotide changes. Genome editing tools are applicable to precise gene correction. Development of genome editing tools comes in two waves. The first wave focuses on improving targeting specificity and editing efficiency of nucleases, and the second wave of gene editing draws on innovative engineering of fusion proteins combining deactivated nucleases and other enzymes that are able to create limitless functional molecular tools. This gene editing advancement is going to impact medicine, particularly in hereditary cancers. In this review, we discuss the application of gene editing as an early intervention and possible treatment for hereditary cancers, by highlighting a selection of highly penetrant cancer syndromes as examples of how this may be achieved in clinical practice.
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The Wisdom of Germline Editing: An Ethical Analysis of the Use of CRISPR-Cas9 to Edit Human Embryos.
With recent reports that a Chinese scientist used CRISPR-Cas9 to heritably edit the genomes of human embryos (i.e., germline editing) brought to term, discussions regarding the ethics of the technology are urgently needed. Although certain applications of germline editing have been endorsed by both the National Academy of Sciences (US) and the Nuffield Council (UK), this paper explores the ethical concerns related even to such therapeutic uses of the technology. Additionally, this paper questions whether the technology could ever feasibly be contained to the therapeutic realm. Consequently, this paper necessarily considers the ethical concerns related to enhancement uses of the technology even if only therapeutic applications are initially considered. In light of the concomitant risks, this paper assesses the technology's countervailing benefits to conclude they do not prevail given that similar outcomes can largely be achieved with existing technologies. Consequently, this paper recommends an international ban on germline editing.
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Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition.
Cas12a is an RNA-guided endonuclease that has been widely used for convenient multiplex gene editing with low off-target effects. To minimize off-targeting in gene editing, we engineered a variant of LbCas12a (termed Lb-K538R) with more stringent PAM recognition, lower off-targeting capability, and similar editing efficiency in vivo compared with LbCas12a. We also demonstrated that Lb2Cas12a from Lachnospiraceae bacterium MA2020 has extensive gene-editing activities in mammalian cells. Similar to Lb-K538R, the designed Lb2Cas12a variant (termed Lb2-K518R) not only had a more stringent PAM sequence change from YYN to TYN (Y is T or C, N is A, T, C, or G), but also displayed lower off-target effects, thereby enabling more potential target site selections with low off-targeting than the common TTTV (V is A, G, or C) PAM. To determine whether this type of mutation at the homologous position had similar effects in other Cas12a, As-K548R was evaluated. Based on the results of the genome-wide off-target test, As-K548R displayed lower off-target effects. Collectively, our findings indicate that the Cas proteins could be designed to be stringent in PAM recognition to reduce their off-target effects, which suggests a promising and practical approach for minimizing off-targets effects in genome editing.
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Genome Editing.
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Genome editing of Spp1 by inhalable CRISPR/Cas9 formulation for treating pulmonary fibrosis.
Secreted phosphoprotein 1 (Spp1) encoding osteopontin (OPN), a matrix cell protein with pro-inflammatory and pro-necrotic tissue properties, plays a crucial role in the onset and progression of idiopathic pulmonary fibrosis (IPF). In order to treat IPF by taking advantage of Spp1, we herein developed an inhalable system composed of calcium phosphate/ poly (lactic-co-glycolic acid) (PLGA) core-shell nanoparticles which are loaded with CRISPR/Cas9 system targeting Spp1 to investigate its therapeutic potential. Specifically, the plasmid encoding Cas9 and single-guide RNA (sgRNA) selectively targeting Spp1 gene was first condensed by calcium phosphate to form Cas9 complexes, which was then encapsulated by PLGA to formulate into a gene-editing inhalable delivery system (termed CaP/Cas9/PLGA). Interestingly, the aerosolized inhaled delivery of CaP/Cas9/PLGA nanoparticles results in the effective traverse of mucosal barriers to fibrotic lungs, where they are internalized by lung cells without inducing noticeable cytotoxicity. Following endo/lysosomal escape and gene expression of CRISPR system, the disruption of Spp1 gene by Cas9/sgRNA induces the mutation frequency exceeding 30 %, resulting in efficient down-regulation of OPN level. In a bleomycin-induced pulmonary fibrosis mouse model, the inhalation of aerosolized CaP/Cas9/PLGA complexes significantly attenuates fibrosis development and improves lung function with undetectable systemic toxicity. This current study defines an innovative inhalable gene-editing formulation and offers a promising gene therapy modality for treating IPF.
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Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish.
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