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A recombineering-based platform for high-throughput genomic editing in Escherichia coli.
UNLABELLED: Functional analysis of bacterial genes or genomic fragments in vivo primarily relies on the analysis of knockout strains. Although various methods have successfully generated bacterial knockout mutants, the parallel operation of multiple sites, especially in biofoundries, remains challenging. New technological refinements of existing methods are necessary for high-throughput genomic editing in bacteria. In this study, to modify numerous sites in parallel, we optimized the linear donor DNA by adding modification at the different positions and achieved high-efficiency recombination with chemical transformation. Then, by combining with the CRISPR system, we established a guide sequence-independent and donor DNA-mediated genomic editing (GIDGE) method, enabling efficient and scarless engineering of common E. coli strains as well as wild-type strains such as E. coli MG1655, with particularly marked advantages demonstrated in E. coli Nissle 1917. This method allows for high-throughput genomic engineering in a 96-well format and is useful for sequence deletion with a wide range of lengths, sequence insertion, sequence replacement, and point mutation. As a proof-of-concept study, we constructed 96 single-gene knockout mutants and a genomic large-fragment deletion library in E. coli K-12 MG1655 using the GIDGE method. This high-throughput and easy-to-use method has great potential for automation and can be adapted for use in biofoundries. IMPORTANCE: With the increasing demand in the microbiology field and the expansion of its application scope, the urgency for genome editing techniques that are not only efficient and versatile but also capable of high-throughput processing and even automation has become increasingly critical. In this study, we enhanced the efficiency of recombination engineering by incorporating modifications and integrated it with the CRISPR system to develop an advanced gene editing method. This method allows for various gene editing events such as insertion, replacement, and long fragment knockout without the need for plasmid construction. It not only demonstrated high efficiency in common E. coli strains but also exhibited marked advantages in the probiotic strain E. coli Nissle 1917. This method is a versatile, efficient approach capable of high-throughput parallel gene editing. Using this method, we successfully constructed a large-scale strain library, significantly accelerating the process of microbial engineering.
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Engineering strategies to safely drive CAR T-cells into the future.
Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.
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CRISPR takes on Huntington's disease.
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A Candidozyma (Candida) auris-Optimized Episomal Plasmid-Induced Cas9-Editing System Reveals the Direct Impact of the S639F-Encoding FKS1 Mutation.
BACKGROUND: Mutations in the Candidozyma (Candida) auris β-glucan synthase gene (FKS1) altering S639 are frequently associated with clinical echinocandin resistance. We have developed a novel C auris-optimized episomal plasmid-induced Cas9 (EPIC) gene-editing system capable of recyclable precision editing and demonstrate the contribution of FKS1S639F mutation to echinocandin resistance. METHODS: The EPIC gene-editing system was generated for optimized use in C auris, and ADE2 modification was evaluated in 5 C auris clades. Mutations leading to Fks1S639F and Fks1WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from clades III and I, respectively. Echinocandin susceptibility was determined by Clinical and Laboratory Standards Institute methods. Cell wall abundance of chitin and β-glucan was assessed by staining with calcofluor white and aniline blue. RESULTS: The EPIC system was capable of targeted ADE2 editing in all tested C auris isolates and precise editing confirmed by sequencing. A single-nucleotide polymorphism (SNP) in FKS1 resulting in either the S639F substitution or a synonymous mutation was introduced in an echinocandin-susceptible clade III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Introduction of the Fks1S639F allele increased echinocandin resistance, while correction of the Fks1S639F to the Fks1WT sequence in an echinocandin-resistant clade I isolate restored echinocandin sensitivity. Evaluation of cell wall composition showed strains harboring Fks1S639F to contain significantly elevated β-glucan and chitin content. CONCLUSIONS: These data demonstrate the potential of our EPIC system in its ability to introduce SNPs in multiple C auris clade backgrounds while revealing the direct impact of the S639F-encoding FKS1 mutation on echinocandin resistance.
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CRISPR-Cas9 Genome Editing and Rapid Selection of Cell Pools.
The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.
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Gene Therapy 2017: Progress and Future Directions.
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Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments.
The CRISPR/Cas9 system has enabled efficient modification of genes in a variety of cellular systems for studying phenotypic effects of genetic perturbations. However, with this technology comes the inherent risk of generating off-target effects (OTEs) in addition to the desired modifications. As such, it can be difficult to conclusively determine that the observed phenotypic changes are in fact due to the intended modification of the target gene and not from random mutations elsewhere in the genome. In addition, biological variations observed within cultured cells or laboratory animals can also confound results and need to be addressed. In this article, we review potential sources of experimental and biological variation as well as propose experimental options to minimize and control OTEs and other variations in CRISPR genome editing experiments for exploratory research applications. Confirmation of on-target KO effect by orthogonal approaches is also discussed.
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CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.
Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.
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Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris.
The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.
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Development of fragrant broomcorn millet (Panicum miliaceum L.) via CRISPR/Cas12i.3-mediated genome editing.
Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.
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