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CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.
Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.
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CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.
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Efficient genome editing of differentiated renal epithelial cells.
Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.
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30 Years of Safe Genetic Engineering in Germany.
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In vivo genome editing thrives with diversified CRISPR technologies.
Prokaryotic type II adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in site-specific genome editing and has revolutionized biomedical research due to its superior efficiency and flexibility. Recent studies have greatly diversified CRISPR technologies by coupling it with various DNA repair mechanisms and targeting strategies. These new advances have significantly expanded the generation of genetically modified animal models, either by including species in which targeted genetic modification could not be achieved previously, or through introducing complex genetic modifications that take multiple steps and cost years to achieve using traditional methods. Herein, we review the recent developments and applications of CRISPR-based technology in generating various animal models, and discuss the everlasting impact of this new progress on biomedical research.
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A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles.
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
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CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002.
Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.
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Convergence of nanotechnology and CRISPR-based diagnostics.
In addition to its broad application in genome engineering and therapeutics, clustered regularly interspaced short palindromic repeats (CRISPR) technology provides field-deployable methods for the highly sensitive and selective detection of nucleic acids. From a diagnostic perspective, CRISPR-based assays hold clear clinical potential for identifying a range of both infectious and non-communicable diseases. In this Perspective we evaluate recent nanotechnologies and nanomaterials that have been engineered to interface with CRISPR systems on a nanoscale level to realize the full potential of this versatile diagnostic tool. We assess biomolecules such as enzymes and oligonucleotides, some of the more commonly used synthetic nanoparticles and detection platforms that integrate nanotechnologies in new and innovative ways. We discuss current trends and look ahead to future challenges and opportunities, including non-nucleic acid target detection, pre-amplification-free detection of nucleic acids, the development of wearable devices and integration with artificial intelligence workflows.
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Gene Editing and CRISPR Therapeutics: Strategies Taught by Cell and Gene Therapy.
A few years ago, we assisted in the demonstration for the first time of the revolutionary idea of a type of adaptive-immune system in the bacteria kingdom. This system, named CRISPR, and variants engineered in the lab, have been demonstrated as functional with extremely high frequency and fidelity in almost all eukaryotic cells studied to date. The capabilities of this RNA-guided nuclease have added to the interest that was announced with the advent of previous technologies for genome editing tools, such as ZFN and TALEN. The capabilities exhibited by these gene editors, opens up a novel scenario that indicates the promise of a next-generation medicine based on precision and personalized objectives, mostly due to the change in the paradigm regarding gene-surgery. This has certainly attracted, like never before, the attention of the biotech business and investor community. This chapter offers a brief overview of some of the factors that have contributed to a rapid entry into the biotech and pharmaceutical company's pipeline, focusing on how cell and gene therapies (CGT), collectively known as advanced therapies, have become the driving forces toward the therapeutic uses of gene editing technology. The sum of all those efforts for more than 30years has contributed to the new paradigm of considering genes as medicines.
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High throughput, efficacious gene editing & genome surveillance in Chinese hamster ovary cells.
Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Furthermore, we validate sequence decomposition as a cost effective, rapid, and accurate method for assessing mutants and eliminating non-clonal CHO populations using only capillary sequencing.
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