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Abstract Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.

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Marine bio-resources are being extensively researched as a priceless supply of substances with therapeutic potential. This work report the first time attempt made towards the green synthesis of gold nanoparticles (AuNPs) using the aqueous extract of marine soft coral (SCE), Sarcophyton crassocaule. The synthesis was conducted under optimized conditions and the visual coloration of reaction mixture changed from yellowish to ruby red at 540 nm. The electron microscopic (TEM, SEM) studies exhibited spherical and oval shaped SCE-AuNPs in the size ranges of 5–50 nm. The organic compounds present in SCE were primarily responsible for the biological reduction of gold ions validated by FT-IR while the zeta potential confirmed the overall stability of SCE-AuNPs. The synthesized SCE-AuNPs exhibited variety of biological efficacies like antibacterial, antioxidant and anti-diabetic in nature. The biosynthesized SCE-AuNPs demonstrated remarkable bactericidal efficacy against clinically significant bacterial pathogens with inhibition zones of mm. Additionally, SCE-AuNPs exhibited greater antioxidant capacity in terms of DPPH: 85 ± 0.32% and RP: 82 ± 0.41%). The ability of enzyme inhibition assays to inhibit α-amylase (68 ± 0.21%) and α-glucosidase (79 ± 0.2%) was quite high. The study also highlighted the spectroscopic analysis of the biosynthesized SCE-AuNPs' catalytic effectiveness of 91% in the reduction processes of the perilous organic dyes, exhibiting pseudo-first order kinetics.

In the current study, aqueous extract of O. scolopendrina (OSE) was used to synthesize AgNPs in a simple and environmentally friendly manner. The biosynthesized OSE-AgNPs were also assessed for its catalytic, antibacterial, anti-diabetic, antioxidant and dye degradation properties. The techniques like UV–visible spectroscopic examinations, TEM, SEM, TGA, zeta potential and FT-IR were used in the characterization investigations. The bioproduction of OSE-AgNPs was preliminary confirmed by UV–visible spectroscopic based investigation followed by microscopic visualization. The synthesized OSE-AgNPs exhibited a reddish brown colour and nearly spherical forms with sizes between 5 and 50 nm quantified by TEM and SEM. The attendance of functional groups like –OH and –NH present in OSE caps on the AgNPs surface was confirmed by FTIR analysis. Interestingly, in the presence of OSE-AgNPs, the degradation of dyes (CV, 95% and EY, 96% in 15 min) were noticeably accelerated. Further, OSE-AgNPs demonstrated substantial antibacterial activity; robust antioxidant properties andnotable anti-diabetic activities. This is the first account on the biosynthetic process of AgNPs using the aqueous extract of O. scolopendrina.

Underwater acoustic (UWA) communication is the only option for underwater long-distance wireless communication. However, its data rate is very low, in tens of kbps, and the time-varying nature of the UWA channel exhibits a Doppler effect. The data rate can be increased using a multicarrier technique, such as Filter Bank Multicarrier (FBMC). The FBMC-OQAM system does not require a cyclic prefix (CP) to eliminate intersymbol interference (ISI). Therefore, compared to other multicarrier techniques, the FBMC-OQAM system achieves the maximum spectral efficiency. Despite the well-designed filter, synchronisation errors cause performance degradation in the FBMC-OQAM system. In this paper, we propose frame boundary and Doppler frequency estimation methods for the FBMC-OQAM system under UWA channels. In this study, we propose a preamble-based frame boundary. The conjugate symmetry property is used in the frame boundary estimation. The performance of the proposed technique was studied under different UWA channels with different Doppler scale. The proposed time synchronisation technique yielded multiple peaks under UWA channels. A method for identifying the peak that represents the frame boundary is proposed. We compared the performance of proposed timing metric with existing three reference timing metrics. The proposed timing metric exhibited superior over two metrics and almost identical performance with the Liming’s metric with less computational complexity. In addition, we propose a convolutional neural network (CNN)-based Doppler scale estimation method. The proposed technique was shown to estimate a wide range of Doppler scale factors. The performance of the proposed technique was studied by considering different UWA channels at different SNRs. The proposed CNN architecture is analysed by changing the CNN parameters. Stacked autoencoders were adopted for Doppler scale estimation and the performance of the proposed CNN-based Doppler scale estimation method was compared with the stacked autoencoders based Doppler scale estimation method. Simulation results show that the proposed CNN-based Doppler Scale estimator exhibits good performance in UWA channels at different SNRs and Doppler scale factors.

Background: Children with disabilities are expected to have poor nutritional status in comparison to children without disabilities. However, limited data on nutritional status of children with and without disabilities in rural settings in India. Objective: To assess and compare the nutritional status of children with and without disability. Methods: A cross-sectional study among children aged 5–15 years was conducted in the rural practise area of a medical college in Karnataka. 290 children (145 with and 145 children without disability) of similar age and sex were studied. Age and sex-specific World Health Organization (WHO) BMI centiles, 24 h dietary calorie and protein intakes were assessed and compared. Median and interquartile ranges were calculated for quantitative variables. Mann–Whitney U test was used to assess the differences in quantitative variables among the two groups. Results: As per WHO BMI centiles, 33.1% with and 37.20% without disabilities were undernourished. The median calorie consumed by children with disabilities was 1169.0 (946.5–1586.0) significantly lower compared to that of children without disability, that is, 1362.0 (1167.0–1641.0). The median protein consumed by children with disabilities was 28.0 (22.5–38.0) significantly lower compared to that of children without disability, that is, 32.0 (28.0–40.0). Conclusions: Children with disabilities had similar rates of undernutrition as that of their non-disabled peers and their lesser dietary intake in terms of calories and proteins.

Abstract Background Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR–Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout. Results In this study, we report for the first time an alternative approach for CRISPR–Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines. Conclusion We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Green chemistry has paved an ‘avant-garde avenue’ in the production and fabrication of eco-friendly stable nanoparticles employing the utilization of biological agents. In the present study we present the first report on the potential of the marine bacterium Lysinibacillus odysseyi PBCW2 for the extracellular production of gold nanoparticles (AuNPs). Utilizing a variety of methods, AuNPs in the cell-free supernatant of L. odysseyi (CFS-LBOE) were identified and their antioxidant, antibacterial, and dye-degrading properties were examined. The visual coloring of the reaction mixture to a ruby red hue showed the production of LBOE-AuNPs; validated by means of XRD, TEM, SEM, XRD, DLS, TGA, and FT-IR analysis. Additionally, the 2,2-diphenyl-1-picrylhydrazyl technique and the well diffusion assay were used to examine their dose-dependent antioxidant and antibacterial activity. These biogenic LBOE-AuNPs showed 91% dye degradation efficiency during catalytic reduction activity on BTB dye, demonstrating their versatility as options for heterogeneous catalysis.

CRISPR-Cas9 genome engineering tools have found wide application in a range of species, however they are unsuitable for applications at elevated temperatures. Here the authors characterise ThermoCas9 from which is functional from 20°C to 70°C.

Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, “tambjamines”, produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates’ ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications.

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

Seaweeds of the intertidal regions are a rich source of surface associated bacteria and are potential source of antimicrobial molecules. In the present study, 77 epiphytic isolates from eight different algae collected from Little Andaman were enumerated. On testing for their antimicrobial activities against certain pathogens twelve isolates showed positive and six of them showed significant antimicrobial inhibition zone against Shigella boydii type 1, Shigella flexneri type 2a, Shigella dysenteriae type 5, Enterotoxigenic Escherichia coli O115, Enteropathogenic E. coli serotype O114, Vibrio cholera; O1 Ogawa, Aeromonas hydrophila, Klebsiella pneumoniae, Staphylococcus aureus. Based on the activity these six isolates (G1C, G2C, G3C, UK, UVAD, and Tor1) were identified by 16S rRNA gene sequence and were found to belong to the phyla Firmicutes and Proteobacteria. Purified antimicrobial compounds obtained from these isolates were identified by GC-MS. Furan derivatives were identified from G2C Pseudomonas stutzeri KJ849834, UVAD Alcanivorax dieselolei KJ849833, UK Vibrio sp. KJ849837, Tor1 Exiguobacterium profundum KJ849838. While 2-Pyrrolidinone, Phenol, 2, 4-bis (1, 1-dimethylethyl) were from G3C Vibrio owensii KJ849836 and (1-Allylcyclopropyl) methanol from the extracts of G1C Bacillus sp. KJ849835. The results of the present study shows that these six potent isolates isolated from the seaweeds are found to be a source of antimicrobial compounds.

Opisthobranchs are the least studied group of marine gastropod molluscs in India. They are purely marine animals and display a wide array of colours and forms. This paper presents a new record of an opisthobranch, Phanerophthalmus smaragdinus, from Andaman Islands. The species was found inhabiting the intertidal area on rocks covered with green and brown algae.

Abstract: Concentration levels of Mn, Pb, Zn, Cd, Cu and Cr in six seaweed samples (Acetabularia calyculus, Corallina sp., Galaxura marginata, Sargassum duplicatum, Sargassum swartzi and Dictyota bartayresiana) were determined from Wandoor, south Andaman Island. Metals were extracted from sample homogenates and quantified by atomic absorption spectrometry. In the present investigation, heavy metal concentration levels in the following order: Mn>Pb>Cd. It is observed that Zn was only present in free floating brown seaweed S. swartzi. Cu and Cr did not show its presence in any of the seaweeds that was sampled. Metal pollution index (MPI) of six seaweed species were observed in the following decending order: A. calyculus > Corallina sp.> D. bartayresiana > G. marginata > S. duplicatum > S. swartzi. Results showed that chlorophyta, A. calyculus contained the highest concentration of heavy metals as compared to other algal species. One-way analysis of variance (ANOVA) showed that the concentration of metals was significantly different (p <0.05) with respect to different species around the study area. [Copyright &y& Elsevier]

The diversity, distribution, and mechanisms of bacterial speciation of Vibrio species belonging to Harveyi clade are an important global research interests due to their pathogenic activity in coastal environments. Luminous bacteria are also known to act as environmental indicators in coastal waters. This study demonstrates that luminous bacteria belonging to harveyi clade are predominant in seawater, sediment, surfaces of marine animals and plants, and light organs of leiognathid fishes. Molecular phylogenies for eighteen morphologically distinct and potentially luminous strains chosen out of 57 isolated luminous bacteria. Sequence analysis of luxA gene as a molecular marker identified luminous bacteria belonging to Harveyi clade, Photobacterium clade, and Anguillarum clade distinctly. Rich biodiversity and distribution of luminous bacterial species (30% to 40%) was found in association with coral reef samples of south Andaman. This study confirms and reveals the evidence on predominant association of Harveyi clade luminous vibrio's in coastal waters of south Andaman. • We show the dispersal of luminous bacterial species in atypical habitats in Andaman waters. • Harveyi clade luminous members were predominant in Andaman waters. • Coral reef biota colonized high number of luminous bacteria than other samples. • Luminous bacteria can be used as pollution indicators in coastal waters. [ABSTRACT FROM AUTHOR]

TnpB proteins are transposon-encoded nucleases involved in transposon DNA propagation. Wiegand et al. identified a new class of TnpB-derived proteins, called TnpB-like nuclease-dead repressors (TldRs), which function as RNA-guided transcriptional regulators targeting conserved promoter regions. In Enterobacteriaceae, bacteriophages use TldRs and an adjacent phage gene to modulate host flagellar assembly., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic and diagnostic landscape. However, CRISPR-Cas9, which edits DNA by activating DNA double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation repair is required. Prime editing, the latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without the requirement for DSBs. However, prime editing is still in its infancy, and further development is needed to improve editing efficiency and delivery strategies for therapeutic applications. We summarize latest developments in the optimization of prime editor (PE) variants with improved editing efficiency and precision. Moreover, we highlight some potential therapeutic applications., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window., Competing Interests: Declaration of interests Two patent applications have been filed related to this work, with J.v.d.O., P.M., W.Y.W., and S.C.A.C. as inventors. J.v.d.O. is founder and scientific adviser of NTrans Technologies and adviser of Scope Biosciences and Hudson River Biotechnology. W.X.Y. and D.A.S. are employees and shareholders of Arbor Biotechnologies, Inc. C.L.B. is a co-founder and member of the scientific advisory board for Locus Biosciences and is a member of the scientific advisory board for Benson Hill. C.L.B. has submitted patent applications on CRISPR technologies unrelated to this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Francisella novicida Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium Bacillus smithii as our experimental platform, we demonstrate that FnCas12a is active in vivo at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for B. smithii , combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of B. smithii cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of Chitinophaga and Flavobacterium was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from Flavobacterium was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools., (© FEMS 2019.)

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli . It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli ., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications., (Copyright © 2016, American Association for the Advancement of Science.)