Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.

New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

RNA-mediated Allosteric Activation in CRISPR-Cas13a.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity.

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTS: Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches.Understanding Cas13a's allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants.These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains.

Aerosol Jet Printing-Enabled Dual-Function Electrochemical and Colorimetric Biosensor for SARS-CoV-2 Detection.

An aerosol jet printing-enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13:guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli.

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His 10 fusion in Escherichia coli . The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.

Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli.

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10 fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.

Aerosol Jet Printing Enabled Dual-Function Electrochemical and Colorimetric Biosensor for SARS-CoV-2 Detection.

An aerosol jet printing enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13: guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

Gold Nanoparticle Enabled Localized Surface Plasmon Resonance on Unique Gold Nanomushroom Structures for On-Chip CRISPR-Cas13a Sensing.

A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.

Optimization of specific RNA knockdown in mammalian cells with CRISPR-Cas13.

Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas systems contain a single effector ribonuclease, Cas13, that binds and processes a CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can be engineered to target and modulate RNA processes in human cells, illustrating the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, editing, or imaging tool [6-8]. While Cas13 has been successfully used by several groups, our lab has observed significant variability in Cas13 KD ability depending which protocol is being followed [9-12]. To further understand this variability and generate a robust Cas13 KD protocol we thoroughly tested which Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA transfected, methods for analyzing KD efficiency, and report an optimized method for carrying out and analyzing Cas13 mediated RNA KD experiments. The method outlined in this paper illustrates a faster and more reliable protocol to iteratively test gRNA performance and target gene KD.

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues.

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.

THUMPD1 bi-allelic variants cause loss of tRNA acetylation and a syndromic neurodevelopmental disorder.

Covalent tRNA modifications play multi-faceted roles in tRNA stability, folding, and recognition, as well as the rate and fidelity of translation, and other cellular processes such as growth, development, and stress responses. Mutations in genes that are known to regulate tRNA modifications lead to a wide array of phenotypes and diseases including numerous cognitive and neurodevelopmental disorders, highlighting the critical role of tRNA modification in human disease. One such gene, THUMPD1, is involved in regulating tRNA N4-acetylcytidine modification (ac4C), and recently was proposed as a candidate gene for autosomal-recessive intellectual disability. Here, we present 13 individuals from 8 families who harbor rare loss-of-function variants in THUMPD1. Common phenotypic findings included global developmental delay, speech delay, moderate to severe intellectual deficiency, behavioral abnormalities such as angry outbursts, facial dysmorphism, and ophthalmological abnormalities. We demonstrate that the bi-allelic variants identified cause loss of function of THUMPD1 and that this defect results in a loss of ac4C modification in small RNAs, and of individually purified tRNA-Ser-CGA. We further corroborate this effect by showing a loss of tRNA acetylation in two CRISPR-Cas9-generated THUMPD1 KO cell lines. In addition, we also show the resultant amino acid substitution that occurs in a missense THUMPD1 allele identified in an individual with compound heterozygous variants results in a marked decrease in THUMPD1 stability and RNA-binding capacity. Taken together, these results suggest that the lack of tRNA acetylation due to THUMPD1 loss of function results in a syndromic form of intellectual disability associated with developmental delay, behavioral abnormalities, hearing loss, and facial dysmorphism.

Put on Your Para-spectacles: The Development of Optimized CRISPR-Cas13-Based Approaches to Image RNA Dynamics in Real Time.

Live-cell RNA imaging is a powerful approach to observe the real-time dynamics of RNA metabolism. Two recent papers describe an optimized fluorescence-based CRISPR-Cas13 approach to image colocalized or repeat-containing RNAs in real time, as well as demonstrate simultaneous RNA-DNA labeling by using Cas13 and Cas9 in tandem.

Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR-Cas Systems.

Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, contain a single protein, Cas13 (formerly C2c2) that when assembled with a CRISPR RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, is required for degradation of target-RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications.

Engineering RNA-Binding Proteins by Modular Assembly of RanBP2-Type Zinc Fingers.

Deciphering the function of the nonprotein-coding portion of genomes represents one of the major challenges that molecular biology is facing today. Numerous classes of RNAs have been discovered over the last past decade and appear to play important regulatory roles in gene expression and disease. The ability to study and manipulate these RNAs relies on the development of programmable RNA-binding molecules such as RNA-binding proteins. Most RNA-binding proteins have modular architectures and combine different RNA-binding domains that provide binding affinity toward a specific RNA sequence and/or structure. Herein, we describe a general strategy to design single-stranded RNA-binding proteins using RanBP2-type zinc-finger (ZF) domains that can recognize a given RNA sequence with high affinity and specificity.

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a.

CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.

RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes.

CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications.