CRISPR System Research Articles

Optimal methods for analyzing targeted pairwise knockout screens.

Synthetic lethality offers a promising strategy for cancer treatment by targeting genetic vulnerabilities unique to tumor cells, leading to selective tumor cell death. However, single-gene knockout screens often miss functional redundancy due to paralog genes. Multiplex CRISPR systems, including various Cas9 and Cas12a platforms, have been developed to assay genetic interactions, yet no systematic comparison of method to identify synthetic lethality from CRISPR screens has been conducted. We evaluated data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using three bioinformatic approaches to identify synthetic lethal interactions: delta log fold change (dLFC), Z-transformed dLFC (ZdLFC), and rescaled dLFC (RdLFC). Both ZdLFC and RdLFC provided more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method. The ZdLFC method offers a robust framework for scoring synthetic lethal interactions from paralog screens, providing consistent results across different cell lines without requiring a training set of known positive interactors.

Versatile and efficient mammalian genome editing with Type I-C CRISPR System of Desulfovibrio vulgaris.

CRISPR-Cas tools for mammalian genome editing typically rely on single Cas9 or Cas12a proteins. While type I CRISPR systems in Class I may offer greater specificity and versatility, they are not well-developed for genome editing. Here, we present an alternative type I-C CRISPR system from Desulfovibrio vulgaris (Dvu) for efficient and precise genome editing in mammalian cells and animals. We optimized the Dvu type I-C editing complex to generate precise deletions at multiple loci in various cell lines and pig primary fibroblast cells using a paired PAM-in crRNA strategy. These edited pig cells can serve as donors for generating transgenic cloned piglets. The Dvu type I-C editor also enabled precise large fragment replacements with homology-directed repair. Additionally, we adapted the Dvu-Cascade effector for cytosine and adenine base editing, developing Dvu-CBE and Dvu-ABE systems. These systems efficiently induced C-to-T and A-to-G substitutions in human genes without double-strand breaks. Off-target analysis confirmed the high specificity of the Dvu type I-C editor. Our findings demonstrate the Dvu type I-C editor's potential for diverse mammalian genome editing applications, including deletions, fragment replacement, and base editing, with high efficiency and specificity for biomedicine and agriculture.

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Developing guanine base editors for G-to-T editing in rice.

Two guanine base editors created using an engineered N-methylpurine DNA glycosylase with CRISPR systems achieved targeted G-to-T editing with 4.94-12.50% efficiency in rice (Oryza sativa). The combined use of the DNA glycosylase and deaminases enabled co-editing of target guanines with adenines or cytosines.

Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.

Safety assessment of probiotic Lactiplantibacillus plantarum MCC5231 and its persistence in gastrointestinal tract

Probiotics are the health beneficial microorganisms and suitable for food industry if found fit for human consumption. In the present study, Lactiplantibacillus plantarum MCC5231, a probiotic bacterium included in vegetable-based beverages, was evaluated for its safety characteristics and gastrointestinal survival using a combined in silico and in vitro approach. The strain was found to be devoid of hemolytic, lecithinase and gelatinase activities. Additionally, it does not consist any transferable antibiotic resistance genes. Further, whole genome sequence analysis revealed the presence of three intact prophages and 14 virulence-associated genes, however, none of them posed a pathogenic threat. Importantly, MCC5231 do not possess any gene associated with toxin production. The strain harbored a CRISPR system, enhancing defense against prophages. Survival assays under simulated gastric and intestinal fluid conditions demonstrated viability rates of 71.4 % and 83.3 %, respectively. Genetic analysis of the mucin binding protein indicated possession of a type II mucin binding domain, suggesting moderate adhesion to intestinal cells. Furthermore, L. plantarum MCC5231 exhibited the ability to produce exopolysaccharides and form biofilms, which may confer additional protection in the gastrointestinal tract. Based on these findings, L. plantarum MCC5231 appears to be a safe probiotic candidate suitable for commercial use in the food industry.

Miniature CRISPR-Cas12 Systems: Mechanisms, Engineering, and Genome Editing Applications.

The therapeutic application of CRISPR-based gene editing technology is hindered by the delivery challenges of large Cas nucleases. The emergence of miniature editing tools derived from type V CRISPR systems and their ancestor TnpB nucleases presents promising solutions to counter these obstacles. Notably, the type V CRISPR-Cas12f and -Cas12n systems exhibit not only a concise gene size but also remarkable precision in targeted editing, thereby underscoring their potential as supreme gene editing tools. Although both systems are considered as intermediates in the evolution of TnpB to mature Cas12 effectors, they exhibit distinct biochemical and structural characteristics, demonstrating the diversity and complexity of TnpB's evolutionary outcomes. The diverse evolutionary branches indicate the existence of numerous unexplored compact CRISPR systems in nature, the mining and development of which could potentially revolutionize gene manipulation techniques and pave the way for innovative applications in gene therapy. In this Account, we summarize the recent advances from our group with the research and development of Cas12f and Cas12n genome editing systems, including the identification, characterization, and engineering for improving the editing efficiency. Additionally, we discuss the evolutionary process of the ancestral nuclease TnpB growing into various type V CRISPR systems, giving insight into the discovery of novel compact gene editing systems.

When size matters: A novel compact Cas12a variant for in vivo genome editing.

A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.

Genomic characterisation of generic Escherichia coli from food-producing animals and products of animal origin in South Africa

Escherichia coli is found in diverse environmental niches, including meat and meat products, and is known for its significance in both food safety and public health. In South Africa, whole genomic sequencing (WGS) efforts for E. coli are overwhelmingly skewed toward human isolates with limited studies conducted on non-human isolates. Therefore, the aim of this study was to use WGS to characterise generic strains of E. coli isolated from animal specimens, meat, and meat-based products in South Africa. Based on WGS analysis, a total of 35 E. coli strains were grouped into five phylogroups (A, B1, B2, C, and E), with A (46%) being the most predominant. Virulence-associated genes identified the isolates as either extra-intestinal pathogenic E. coli (69%) or intestinal pathogenic E. coli (31%) pathotypes. Twenty-three different serotypes were identified, with O101:H37 (17%), O2:H4 (17%), O6:H5 (13%), and O64:H19 (9%) being the predominant ones. Among the 19 different sequence types (STs), ST1858, ST975, and ST10 were the most prevalent (11% each). Various virulence genes, antimicrobial resistance genes, and genetic mobile elements carrying Tn2, IS26, and Tn6196 elements were detected, with the disinfectant resistance sitABCD being the most predominant. The type 1 CRISPR system which functions by storing records of previous invasions to provide immunological memory for a rapid and robust response upon subsequent viral infections was detected in all isolates, consisting of subtypes I-E (86%), I-A (57%), and I-F (11%). The findings of this study provide an insight into the genetic diversity of generic E. coli isolates from animal species, meat, and meat-based products in South Africa.

Mechanisms of regeneration: to what extent do they recapitulate development?

One of the enduring debates in regeneration biology is the degree to which regeneration mirrors development. Recent technical advances, such as single-cell transcriptomics and the broad applicability of CRISPR systems, coupled with new model organisms in research, have led to the exploration of this longstanding concept from a broader perspective. In this Review, I outline the historical parallels between development and regeneration before focusing on recent research that highlights how dissecting the divergence between these processes can uncover previously unreported biological mechanisms. Finally, I discuss how these advances position regeneration as a more dynamic and variable process with expanded possibilities for morphogenesis compared with development. Collectively, these insights into mechanisms that orchestrate morphogenesis may reshape our understanding of the evolution of regeneration, reveal hidden biology activated by injury, and offer non-developmental strategies for restoring lost or damaged organs and tissues.

Cas9 interaction with the tracrRNA nexus modulates the repression of type II-A CRISPR-cas genes.

Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity.

Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.

Mutations in microRNA-96 (MIR96) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for the MIR96 mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A/+ mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human MIR96 mutations. Because mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by MIR96 mutations.

Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system.

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.

Modern Breeding Strategies and Tools for Durable Late Blight Resistance in Potato.

Cultivated potato (Solanum tuberosum) is a major crop worldwide. It occupies the second place after cereals (corn, rice, and wheat). This important crop is threatened by the Oomycete Phytophthora infestans, the agent of late blight disease. This pathogen was first encountered during the Irish famine during the 1840s and is a reemerging threat to potatoes. It is mainly controlled chemically by using fungicides, but due to health and environmental concerns, the best alternative is resistance. When there is no disease, no treatment is required. In this study, we present a summary of the ongoing efforts concerning resistance breeding of potato against this devastating pathogen, P. infestans. This work begins with the search for and selection of resistance genes, whether they are from within or from outside the species. The genetic methods developed to date for gene mining, such as effectoromics and GWAS, provide researchers with the ability to identify genes of interest more efficiently. Once identified, these genes are cloned using molecular markers (MAS or QRL) and can then be introduced into different cultivars using somatic hybridization or recombinant DNA technology. More innovative technologies have been developed lately, such as gene editing using the CRISPR system or gene silencing, by exploiting iRNA strategies that have emerged as promising tools for managing Phytophthora infestans, which can be employed. Also, gene pyramiding or gene stacking, which involves the accumulation of two or more R genes on the same individual plant, is an innovative method that has yielded many promising results. All these advances related to the development of molecular techniques for obtaining new potato cultivars resistant to P. infestans can contribute not only to reducing losses in agriculture but especially to ensuring food security and safety.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes.

ConspectusSophisticated genetic networks play a pivotal role in orchestrating cellular responses through intricate signaling pathways across diverse environmental conditions. Beyond the inherent complexity of natural cellular signaling networks, the construction of artificial signaling pathways (ASPs) introduces a vast array of possibilities for reshaping cellular responses, enabling programmable control of living organisms. ASPs can be integrated with existing cellular networks and redirect output responses as desired, allowing seamless communication and coordination with other cellular processes, thereby achieving designable transduction within cells. Among diversified ASPs, establishing connections between originally independent endogenous genes is of particular significance in modifying the genetic networks, so that cells can be endowed with new capabilities to sense and deal with abnormal factors related to differentiated gene expression (i.e., solve the issues of the aberrant gene expression induced by either external or internal stimuli). In a typical scenario, the two genes X and Y in the cell are originally expressed independently. After the introduction of an ASP, changes in the expression of gene X may exert a designed impact on gene Y, subsequently inducing the cellular response related to gene Y. If X represents a disease signal and Y serves as a therapeutic module, the introduction of the ASP empowers cells with a new spontaneous defense system to handle potential risks, which holds great potential for both fundamental and translational studies.In this Account, we primarily review our endeavors in the construction of RNA-mediated ASPs between endogenous genes that can respond to differentiated RNA expression. In contrast to other molecules that may be restricted to specific pathways, synthetic RNA circuits can be easily utilized and expanded as a general platform for constructing ASPs with a high degree of programmability and tunability for diversified functionalities through predictable Watson-Crick base pairing. We first provide an overview of recent advancements in RNA-based genetic circuits, encompassing but not limited to utilization of RNA toehold switches, siRNA and CRISPR systems. Despite notable progress, most reported RNA circuits have to contain at least one exogenous RNA X as input or one engineered RNA Y as a target, which is not suitable for establishing endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, constructing a general and efficient platform for manipulation of naturally occurring RNAs poses a formidable challenge, especially for the mammalian system. With a focus on this goal, we are devoted to developing efficient strategies to manipulate cell responses by establishing RNA-mediated ASPs between endogenous genes, particularly in mammalian cells. Our step-by-step progress in engineering customized cell signaling circuits, from bacterial cells to mammalian cells, from gene expression regulation to phenotype control, and from small RNA to long mRNA of low abundance and more complex secondary structures, is systematically described. Finally, future perspectives and potential applications of these RNA-mediated ASPs between endogenous genes are also discussed.

Downregulation of Ripk1 and Nsf mediated by CRISPR-CasRx ameliorates stroke volume and neurological deficits after ischemia stroke in mice.

Necroptosis is implicated in the pathogenesis of ischemic stroke. However, the mechanism underlying the sequential recruitment of receptor-interacting protein kinase 1 (RIPK1) and N-ethylmaleimide-sensitive fusion ATPase (NSF) in initiating necroptosis remains poorly understood, and the role of NSF in ischemic stroke is a subject of controversy. Here, we utilized a recently emerging RNA-targeting CRISPR system known as CasRx, delivered by AAVs, to knockdown Ripk1 mRNA and Nsf mRNA around the ischemic brain tissue. This approach resulted in a reduction in infarct and edema volume, as well as an improvement in neurological deficits assessed by Bederson score, RotaRod test, and Adhesive removal test, which were achieved by RIPK1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein signaling pathway involved in neuronal necroptosis. In conclusion, the downregulation of Ripk1 mRNA and Nsf mRNA mediated by CRISPR-CasRx holds promise for future therapeutic applications aimed at ameliorating cerebral lesions and neurological deficits following the ischemic stroke.

Protection of animals against devastating RNA viruses using CRISPR-Cas13s

The intrinsic nature of CRISPR-Cas in conferring immunity to bacteria and archaea has been repurposed to combat pathogenic agents in mammalian and plant cells. In this regard, CRISPR-Cas13 systems have proved their remarkable potential for single-strand RNA viruses targeting. Here, different types of Cas13 orthologs were applied to knockdown foot-and-mouth disease virus (FMDV), a highly contagious disease of a wide variety of species with genetically diverse strains and is widely geographically distributed. Using programmable CRISPR RNAs capable of targeting conserved regions of the viral genome, all Cas13s from CRISPR system type VI (subtype A/B/D) could comprehensively target and repress different serotypes of FMDV virus. This approach has the potential to destroy all strains of a virus as targets the ultra-conserved regions of genome. We experimentally compared the silencing efficiency of CRISPR and RNAi by designing the most effective short hairpin RNAs according to our developed scoring system and observed comparable results. This study showed successful usage of various Cas13 enzymes for suppression of FMDV, which provides a flexible strategy to battle with other animal infectious RNA viruses, an underdeveloped field in the biotechnology scope.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes.It remainsuncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel β-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.