The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Detection of Staphylococcus aureus virulence gene pvl based on CRISPR strip.

Introduction: Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for both hospital-acquired and community-acquired infections. Among its arsenal of virulence factors, Panton-Valentine Leucocidin (PVL) is closely associated with severe diseases such as profound skin infections and necrotizing pneumonia. Patients infected with pvl-positive S. aureus often exhibit more severe symptoms and carry a substantially higher mortality risk. Therefore, it is crucial to promptly and accurately detect pvl-positive S. aureus before initiating protective measures and providing effective antibacterial treatment. Methods: In this study, we propose a precise identification and highly sensitive detection method for pvl-positive S. aureus based on recombinase-assisted amplification and the CRISPR-ERASE strip which we previously developed. Results: The results revealed that this method achieved a detection limit of 1 copy/µL for pvl-positive plasmids within 1 hour. The method successfully identified all 25 pvl-positive and 51 pvl-negative strains among the tested 76 isolated S. aureus samples, demonstrating its concordance with qPCR. Discussion: These results show that the CRISPR-ERASE detection method for pvl-positive S. aureus has the advantages of high sensitivity and specificity, this method combines the characteristics of recombinase-assisted amplification at room temperature and the advantages of ERASE test strip visualization, which can greatly reduce the dependence on professional laboratories. It is more suitable for on-site detection than PCR and qPCR, thereby providing important value for rapid on-site detection of pvl.

Modular vector assembly enables rapid assessment of emerging CRISPR technologies.

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

CRISPRpi: Inducing and Curing Prophage Using the CRISPR Interference.

We present here a CRISPR-interference-based protocol to trigger prophage induction, even for non-inducible prophages. This method can also be used to cure the prophage from the bacterial host. The method is based on silencing of the phage's repressor transcription, thanks to CRISPR interference. Plasmid electroporation is used to bring the CRISPRi system into the bacteria, specifically on a plasmid carrying spacers targeting the prophage repressor. This method enables prophage induction and curation in a week or two with a high efficiency.

Fragmid: A toolkit for rapid assembly and assessment of CRISPR technologies.

The CRISPR toolbox continues to expand at a rapid pace, leaving researchers scrambling to assess the latest tools in their systems of interest. McGee et al.1 have developed a modular assembly platform with standardized and interchangeable components for rapid construction and deployment of novel CRISPR constructs.

Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit.

The epigenome, consisting of chemical modifications to DNA and histone proteins, can alter gene expression. Clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) systems enable precise target gene-specific gene modulation by attaching different "effector" domains to the dCas9 protein to activate or repress specific genes. CRISPR/dCas9-SunTag is an improved system version, allowing more efficient and precise gene activation or repression by recruiting multiple copies of the protein of interest. A CRISPR/dCas9-SunTag-based modular epigenetic toolkit was developed, enabling gene-specific epigenetic architecture modulation. This protocol generated a stable SH-SY5Y cell line expressing the CRISPR/dCas9-SunTag-JARID1A system to study H3K4Me3-mediated promoter regulation at a 200-400 bp of fine resolution. The procedure involved designing sgRNAs, subcloning dCas9-5XGCN4 into pLvx-DsRed, validating epigenetic mark changes with ChIP, and validating gene expression changes with RT-qPCR. This epigenetic toolkit is valuable for researchers to understand the relationship between gene-specific epigenetic modifications and gene expression.

Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides.

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing of some of the oligos revealed a variety of contaminants. Given the wide range of applications of oligos, the impact of oligo cross-contamination varies greatly depending on the field and the experimental method. Incorporating appropriate control experiments in research design can help ensure that the quality of oligo reagents meets the intended purpose. This can also minimize risk depending on the purposes for which the oligos are used.

A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Genome-wide CRISPR screen reveals the synthetic lethality between BCL2L1 inhibition and radiotherapy.

Radiation therapy (RT) is one of the most commonly used anticancer therapies. However, the landscape of cellular response to irradiation, especially to a single high-dose irradiation, remains largely unknown. In this study, we performed a whole-genome CRISPR loss-of-function screen and revealed temporal inherent and acquired responses to RT. Specifically, we found that loss of the IL1R1 pathway led to cellular resistance to RT. This is in part because of the involvement of radiation-induced IL1R1-dependent transcriptional regulation, which relies on the NF-κB pathway. Moreover, the mitochondrial anti-apoptotic pathway, particularly the BCL2L1 gene, is crucially important for cell survival after radiation. BCL2L1 inhibition combined with RT dramatically impeded tumor growth in several breast cancer cell lines and syngeneic models. Taken together, our results suggest that the combination of an apoptosis inhibitor such as a BCL2L1 inhibitor with RT may represent a promising anticancer strategy for solid cancers including breast cancer.

Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays.

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together 'chimeric' chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.