Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida
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Biotechnology & Applied Microbiology↗
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Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom
Training the future synthetic biology workforce requires the opportunity for students to be exposed to biotechnology concepts and activities in secondary education. Detecting Wolbachia bacteria in arthropods using polymerase chain reaction (PCR) has become a common way for secondary students to investigate and apply recombinant DNA technology in the science classroom. Despite this important activity, cutting-edge biotechnologies such as clustered regularly interspaced short palindromic repeat (CRISPR)-based diagnostics have yet to be widely implemented in the classroom. To address this gap, we present a freeze-dried CRISPR-Cas12 sensing reaction to complement traditional recombinant DNA technology education and teach synthetic biology concepts. The reactions accurately detect Wolbachia from arthropod-derived PCR samples in under 2 h and can be stored at room temperature for over a month without appreciable degradation. Here, the reactions are easy-to-use and cost less than $40 to implement for a classroom of 22 students including the cost of reusable equipment. We see these freeze-dried CRISPR-Cas12 reactions as an accessible way to incorporate synthetic biology education into the existing biology curriculum, which will expand biology educational opportunities in science, technology, engineering, and mathematics.
59 BASIC BIOLOGICAL SCIENCES↗
Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants
CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the trade-off that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Furthermore, our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.
59 BASIC BIOLOGICAL SCIENCES↗
Data for Creating Yellow Seed Camelina sativa with Enhanced Oil Accumulation by CRISPR-Mediated Disruption of Transparent Testa 8
Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.
Biofuels↗
Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly
Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe–microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P m , LacI/P lac , and AraC/P BAD ) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10–100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe–microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Furthermore, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.
Bacteria↗
Machine learning predicts new anti-CRISPR proteins
The increasing use of CRISPR–Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR–Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR–Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.
59 BASIC BIOLOGICAL SCIENCES↗
Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences
Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.
59 BASIC BIOLOGICAL SCIENCES↗
Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling
Understanding CRISPR-Cas systems—the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks—is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical ‘rich-get-richer’ mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.
59 BASIC BIOLOGICAL SCIENCES↗
Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex
CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1—Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.
59 BASIC BIOLOGICAL SCIENCES↗
Genome expansion by a CRISPR trimmer-integrase
CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR–Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
59 BASIC BIOLOGICAL SCIENCES↗
Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM
Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR–Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR–Cas systems. The type I-F CRISPR–Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. As such, here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR–Cas system.
59 BASIC BIOLOGICAL SCIENCES↗
Long-term CRISPR locus dynamics and stable host-virus co-existence in subsurface fractured shales
Viruses are the most ubiquitous biological entities on Earth. Even so, elucidating the impact of viruses on microbial communities and associated ecosystem processes often requires identification of unambiguous host-virus linkages-an undeniable challenge in many ecosystems. Subsurface fractured shales present a unique opportunity to first make these strong linkages via spacers in CRISPR-Cas arrays and subsequently reveal complex long-term host-virus dynamics. Here, we sampled two replicated sets of fractured shale wells for nearly 800 days, resulting in 78 metagenomes from temporal sampling of six wells in the Denver-Julesburg Basin (Colorado, USA). At the community level, there was strong evidence for CRISPR-Cas defense systems being used through time and likely in response to viral interactions. Within our host genomes, represented by 202 unique MAGs, we also saw that CRISPR-Cas systems were widely encoded. Together, spacers from host CRISPR loci facilitated 2,110 CRISPR-based viral linkages across 90 host MAGs spanning 25 phyla. We observed less redundancy in host-viral linkages and fewer spacers associated with hosts from the older, more established wells, possibly reflecting enrichment of more beneficial spacers through time. Leveraging temporal patterns of host-virus linkages across differing well ages, we report how host-virus co-existence dynamics develop and converge through time, possibly reflecting selection for viruses that can evade host CRISPR-Cas systems. Together, our findings shed light on the complexities of host-virus interactions as well as long-term dynamics of CRISPR-Cas defense among diverse microbial populations.
59 BASIC BIOLOGICAL SCIENCES↗
Gene editing and CRISPR in the clinic: current and future perspectives
Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.
59 BASIC BIOLOGICAL SCIENCES↗
Functional characterization of diverse type I-F CRISPR-associated transposons
CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.
59 BASIC BIOLOGICAL SCIENCES↗
CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria
With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.
59 BASIC BIOLOGICAL SCIENCES↗
Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2
CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.
60 APPLIED LIFE SCIENCES↗
CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger
CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.
59 BASIC BIOLOGICAL SCIENCES↗
The CRISPR effector Cam1 mediates membrane depolarization for phage defence
Prokaryotic type III CRISPR–Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF–transmembrane helix fusion proteins facilitate the type III CRISPR–Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.
59 BASIC BIOLOGICAL SCIENCES↗