Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons.

CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

"Mmm, Smells like Coffee!": How a Brief Odor Identification Test Could Help to Identify People with Mild Cognitive Impairment and Dementia.

(1) Background: Dementia and mild cognitive impairment (MCI) are still underdiagnosed in the general population. Impaired odor identification has been identified as an early marker of MCI and dementia. We aimed to compare the additional diagnostic value of two odor identification tests to a cognitive screening test in detecting MCI or dementia. (2) Methods: The Sniffin' Sticks odor identification test (SS-OIT), a brief odor identification test (B-OIT) requiring the identification of coffee scent, and the Mini-Mental State Exam (MMSE) were administered to a consecutive series of 174 patients (93 with dementia, 42 with mild cognitive impairment, and 39 without cognitive impairment) referred for neuropsychological testing. (3) Results: Both participants with dementia and with MCI exhibited impairments in odor identification. The SS-OIT and the B-OIT were substantially correlated. Complementing MMSE scores with the SS-OIT or the B-OIT similarly improved the diagnostic accuracy of individuals with dementia and MCI. (4) Conclusions: People with suspected dementia or MCI may already benefit from brief odor identification tests. Although these tests require little additional time, they can notably increase sensitivity for dementia or MCI.

"Try to Build This Bunny as Fast as Possible without Using Red or Pink Bricks": How Simple Assembly Tasks Might Aid in Detecting People with Mild Cognitive Impairment and Dementia.

(1) Background: Dementia and mild cognitive impairment (MCI) are still underdiagnosed in the general population. Impaired odor identification has been identified as an early marker of MCI and dementia. We aim to investigate whether short tasks, in which simple forms must be assembled from single building blocks based on a template or while considering specific re-strictions, could increase the diagnostic quality of established cognitive screening tests in detecting MCI or dementia. (2) Methods: A brief assembly test, where participants had to assemble simple animal shapes from Lego® Duplo® building blocks, the Frontal Assessment Battery, and the Mini-Mental State Exam (MMSE) were administered to a consecutive series of 197 patients (89 with mild dementia, 62 with mild cognitive impairment, and 46 without cognitive impairment) referred for neuropsychological testing. (3) Results: Both participants with dementia and with MCI performed badly in the assembly tasks. The assembly tasks and the Frontal Assessment Battery were substantially correlated. Complementing MMSE scores with the assembly tasks improved the diagnostic accuracy of individuals with dementia and MCI. (4) Conclusions: People with suspected dementia or MCI may already benefit from simple assembly tasks. Although these tests require little additional time, they can notably increase sensitivity for dementia or MCI.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes.

Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.

Cognitive Remediation in Psychiatric Disorders: State of the Evidence, Future Perspectives, and Some Bold Ideas.

Many people with psychiatric disorders experience impairments in cognition. These deficits have a significant impact on daily functioning and sometimes even on the further course of their disease. Cognitive remediation (CR) is used as an umbrella term for behavioral training interventions to ameliorate these deficits. In most but not all studies, CR has proven effective in improving cognition and enhancing everyday functional outcomes. In this paper, after quickly summarizing the empirical evidence, practical advice to optimize the effects of CR interventions is provided. We advocate that CR interventions should be as fun and motivating as possible, and therapists should at least consider using positively toned emotional stimuli instead of neutral stimuli. Participants should be screened for basic processing deficits, which should be trained before CR of higher-order cognitive domains. CR should stimulate metacognition and utilize natural settings to invoke social cognition. Wherever possible, CR tasks should link to tasks that participants face in their everyday life. Therapists should consider that participants might also benefit from positive side effects on symptomatology. Finally, the CR approach might even be utilized in settings where the treatment of cognitive impairments is not a primary target.

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614→Gly) variant with single-base resolution in patient samples.

CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.

CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.