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Advances in CRISPR-enabled genome-wide screens in yeast.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-wide screens are powerful tools for unraveling genotype-phenotype relationships, enabling precise manipulation of genes to study and engineer industrially useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of nonconventional yeast-fast growth rates, high stress tolerance, and novel metabolism-to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering.
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CRISPR links to long noncoding RNA function in mice: A practical approach.
Next generation sequencing has uncovered a trove of short noncoding RNAs (e.g., microRNAs) and long noncoding RNAs (lncRNAs) that act as molecular rheostats in the control of diverse homeostatic processes. Meanwhile, the tsunamic emergence of clustered regularly interspaced short palindromic repeats (CRISPR) editing has transformed our influence over all DNA-carrying entities, heralding global CRISPRization. This is evident in biomedical research where the ease and low-cost of CRISPR editing has made it the preferred method of manipulating the mouse genome, facilitating rapid discovery of genome function in an in vivo context. Here, CRISPR genome editing components are updated for elucidating lncRNA function in mice. Various strategies are highlighted for understanding the function of lncRNAs residing in intergenic sequence space, as host genes that harbor microRNAs or other genes, and as natural antisense, overlapping or intronic genes. Also discussed is CRISPR editing of mice carrying human lncRNAs as well as the editing of competing endogenous RNAs. The information described herein should assist labs in the rigorous design of experiments that interrogate lncRNA function in mice where complex disease processes can be modeled thus accelerating translational discovery.
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Type III CRISPR-mediated flexible RNA excision with engineered guide RNAs.
Current RNA editing techniques are predominantly limited to single-base edits. Here, we introduce selective cleavages and intramolecular stitches of RNA (SCISSOR) for selective cleavage and intramolecular stitches of RNA. Building on the principle that type III CRISPR complex determines target cleavage positions based on gRNA length in 6-nt increments, we hypothesized that engineering gRNAs with bulge loops could circumvent this rule, allowing for flexible RNA excision. Through systematic evaluation of gRNAs with various bulge loops, we established the rules for precise non-6-nt target cleavage and repair. We observed that the complex tolerates 1- or 2-nt bulge loops and accommodates large bulge loops ranging from 6 to 24 nt. Consequently, SCISSOR could accomplish nearly any length of short fragment excision. With its capability to modify open reading frames, we demonstrate the potential of SCISSOR in repairing frameshift mutations and introducing frameshifts to create immunogenic poly-epitopes in human cells. SCISSOR holds promise in RNA therapy and biomedical research.
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CRISPR-mediated gene editing to rescue haploinsufficient obesity syndrome.
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Gene Editing in Sorghum Through Agrobacterium.
The application of CRISPR/Cas to introduce targeted genomic edits is powering research and discovery across the genetic frontier. Applying CRISPR/Cas in sorghum can facilitate the study of gene function and unlock our understanding of this robust crop that serves as a staple for some of the most food insecure regions on the planet. When paired with recent advances in sorghum tissue culture and Agrobacteria technology, CRISPR/Cas can be used to introduce desirable changes and natural genetic variations directly into agriculturally relevant sorghum lines facilitating product development. This chapter describes CRISPR/Cas gene editing and provides high-level strategies and expectations for applying this technology using Agrobacterium in sorghum.
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Multiplex gene editing to promote cell survival using low-pH clustered regularly interspaced short palindromic repeats activation (CRISPRa) gene perturbation.
BACKGROUND AIMS: Lower back pain is the leading cause of disability worldwide and is often linked to degenerative disc disease (DDD), the breakdown of intervertebral discs. The majority of treatment options for DDD are palliative, with clinicians prescribing medication or physical therapy to return the patient to work. Cell therapies are promising treatment options with the potential to restore functional physiological tissue and treat the underlying causes of DDD. DDD is characterized by biochemical changes in the microenvironment of the disc, including changes in nutrient levels, hypoxia, and changes in pH. Stem cell therapies are promising therapies to treat DDD, but the acidic environment in a degenerating disc significantly hinders the viability of stem cells, affecting their efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR) systems allow us to engineer cell phenotypes in a well-regulated and controlled manner. Recently, CRISPR gene perturbation screens have assessed fitness, growth and provided a means for specific cell phenotype characterization. METHODS: In this study, we use a CRISPR-activation (a) gene perturbation screen to identify gene upregulation targets that enhance adipose-derived stem cell survival in acidic culture conditions. RESULTS: We identified 1213 prospective pro-survival genes and systematically narrowed these down to 20 genes for validation. We further narrowed down our selection to the top five prospective genes using Cell Counting Kit-8 cell viability assays in naïve adipose-derived stem cells and ACAN/Col2 CRISPRa upregulated stem cells. Finally, we examined the extracellular matrix-producing abilities of multiplex ACAN/Col2-pro-survival edited cells in pellet culture. CONCLUSIONS: Using the results from the CRISPRa screen, we are able to engineer desirable cell phenotypes to improve cell viability for the potential treatment of DDD and other disease states that expose cell therapies to acidic environments, while also providing broader knowledge on genes regulating low-pH cell survival.
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Gene-edited plants on the plate: the 'CRISPR cabbage story'.
The European Union (EU) has very strict regulations for genetically modified plants, but gene editing challenges the basis of the legislation. Genome editied plants with no foreign DNA added were according to the Swedish competent authority Jordbruksverket falling outside of the EU definition of genetically modified plants, and should hence not be regulated. This article describes the background to this interpretation and also how plants gene edited using CRISPR-Cas9 (a Brassica deletion mutant) for the first (?) time were grown in a garden, cooked and eaten, and the subsequent media attention. Furthermore, it also adresses the troubles authorities in other European countries have to handle these plants.
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Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.
Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.
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CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing.
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CRISPR-Based Technologies for Metabolic Engineering in Cyanobacteria.
In metabolic engineering, the production of industrially relevant chemicals, via rational engineering of microorganisms, is an intensive area of research. One particular group of microorganisms that is fast becoming recognized for their commercial potential is cyanobacteria. Through the process of photosynthesis, cyanobacteria can use CO2 as a building block to synthesize carbon-based chemicals. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-dependent approaches have rapidly gained popularity for engineering cyanobacteria. Such approaches permit markerless genome editing, simultaneous manipulation of multiple genes, and transcriptional regulation of genes. The drastically shortened timescale for mutant selection and segregation is especially advantageous for cyanobacterial work. In this review, we highlight studies that have implemented CRISPR-based tools for the metabolic engineering of cyanobacteria.
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