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Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.
Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.
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DNA interference states of the hypercompact CRISPR-CasΦ effector.
CRISPR-CasΦ, a small RNA-guided enzyme found uniquely in bacteriophages, achieves programmable DNA cutting as well as genome editing. To investigate how the hypercompact enzyme recognizes and cleaves double-stranded DNA, we determined cryo-EM structures of CasΦ (Cas12j) in pre- and post-DNA-binding states. The structures reveal a streamlined protein architecture that tightly encircles the CRISPR RNA and DNA target to capture, unwind and cleave DNA. Comparison of the pre- and post-DNA-binding states reveals how the protein rearranges for DNA cleavage upon target recognition. On the basis of these structures, we created and tested mutant forms of CasΦ that cut DNA up to 20-fold faster relative to wild type, showing how this system may be naturally attenuated to improve the fidelity of DNA interference. The structural and mechanistic insights into how CasΦ binds and cleaves DNA should allow for protein engineering for both in vitro diagnostics and genome editing.
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High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas.
The activity of a conditional guide RNA (cgRNA) is dependent on the presence or absence of an RNA trigger, enabling cell-selective regulation of CRISPR/Cas function. cgRNAs are programmable at two levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, or induce a gene of choice) and the trigger-binding sequence controlling the scope of Cas activity (subset of cells expressing the trigger RNA). Allosteric cgRNA mechanisms enable independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Building on prior advances in dynamic RNA nanotechnology that demonstrated the cgRNA concept, here we set the goal of engineering high-performance allosteric cgRNA mechanisms for the mammalian setting, pursuing both ON → OFF logic (conditional inactivation by an RNA trigger) and OFF → ON logic (conditional activation by an RNA trigger). For each mechanism, libraries of orthogonal cgRNA/trigger pairs were designed using NUPACK. In HEK 293T cells expressing cgRNAs, triggers, and inducing dCas9: (1) a library of four ON → OFF "terminator switch" cgRNAs exhibit a median fold-change of ≈50×, a median fractional dynamic range of ≈20%, and a median crosstalk modulus of ≈9%; (2) a library of three OFF → ON "split-terminator switch" cgRNAs exhibit a median fold-change of ≈150×, a median fractional dynamic range of ≈50%, and a median crosstalk modulus of ≈4%. Further, we demonstrate that xrRNA elements that protect viral RNAs from degradation by exoribonucleases can dramatically enhance the performance of RNA synthetic biology. The high-performance allosteric cgRNAs demonstrated here for ON → OFF and OFF → ON logic in mammalian cells provide a foundation for pursuing applications of programmable cell-selective regulation.
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Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21.
Osteoarthritis (OA) is a highly prevalent disease with limited treatment options. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of biallelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell-derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Furthermore, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis. Impact Statement This work provides two important advances to the field of tissue engineering. One is the demonstration that engineered cartilage tissue can be produced from genetically defined populations of primary human chondrocytes. While CRISPR/Cas-9 genome editing has been extensively used in cell lines that divide indefinitely, this work extends the technique to an engineered tissue model system to support investigation of genetic changes that affect cartilage production. A second contribution is the finding that chondrocytes with p21 knockout synthesized more cartilage matrix tissue than unedited controls. This supports the continued investigation of p21 as a potential barrier to effective cartilage regeneration.
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Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava.
The production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme gene SBE2 was firstly achieved. High-amylose cassava (Manihot esculenta Crantz) is desirable for starch industrial applications and production of healthier processed food for human consumption. In this study, we report the production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme 2 (SBE2). Mutations in two targeted exons of SBE2 were identified in all regenerated plants; these mutations, which included nucleotide insertions, and short or long deletions in the SBE2 gene, were classified into eight mutant lines. Three mutants, M6, M7 and M8, with long fragment deletions in the second exon of SBE2 showed no accumulation of SBE2 protein. After harvest from the field, significantly higher amylose (up to 56% in apparent amylose content) and resistant starch (up to 35%) was observed in these mutants compared with the wild type, leading to darker blue coloration of starch granules after quick iodine staining and altered starch viscosity with a higher pasting temperature and peak time. Further 1H-NMR analysis revealed a significant reduction in the degree of starch branching, together with fewer short chains (degree of polymerization [DP] 15-25) and more long chains (DP>25 and especially DP>40) of amylopectin, which indicates that cassava SBE2 catalyzes short chain formation during amylopectin biosynthesis. Transition from A- to B-type crystallinity was also detected in the starches. Our study showed that CRISPR/Cas9-mediated mutagenesis of starch biosynthetic genes in cassava is an effective approach for generating novel varieties with valuable starch properties for food and industrial applications.
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CRISPR-driven diagnostics: Molecular mechanisms, clinical efficacy and translational challenges.
BACKGROUND: In the realm of public health, among the primary perils menacing human well-being, the issue of pathogen infection persists as a significant concern. Precise and timely diagnosis of diseases constitutes the bedrock for effective therapeutic interventions and epidemiological monitoring. Hence, it is crucial to develop quick, sensitive, and highly effective methods for identifying pathogen and their variants. MATERIAL AND METHODS: This article reviews the recent research progress in the CRISPR/Cas system for detecting nucleic acids, with an emphasis on CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. Initially, we provided a concise overview of the nucleic acid detection mechanism utilizing the CRISPR/Cas system. Subsequently, we dissect the molecular mechanisms of CRISPR tools, compare their clinical efficacy against traditional methods, and explore frontier innovations such as amplification-free detection and AI integration. CONCLUSION: Ultimately, we argue that CRISPR diagnostics must evolve beyond technical optimization to embrace ecological adaptability, ensuring that precision medicine serves as a bridge-rather than a barrier-to global health equity. KEY POINTS: Core Mechanism: Explains the molecular basis of CRISPR-Cas (Cas9, Cas12, Cas13) for nucleic acid detection, leveraging crRNA-guided targeting and trans-cleavage activity for ultra-sensitive (aM level) and specific pathogen identification. Superior Performance: Outperforms traditional methods in speed, sensitivity, and cost, making it ideal for point-of-care use in resource-limited settings. Cutting-Edge Innovations: Covers key advances like amplification-free detection, portable device integration, and multiplex platforms. Translation Challenges: Discusses hurdles in clinical adoption, including inhibitor interference in complex samples, scalability limitations, the need for multi-center clinical data, and varying regional regulations. Future Outlook: Highlights emerging directions such as integrated "sample-to-result" systems and AI integration, while also addressing associated biosafety and ethical concerns, calling for robust regulatory frameworks.
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Structure of the miniature type V-F CRISPR-Cas effector enzyme.
RNA-guided DNA endonucleases derived from CRISPR-Cas adaptive immune systems are widely used as powerful genome-engineering tools. Among the diverse CRISPR-Cas nucleases, the type V-F Cas12f (also known as Cas14) proteins are exceptionally compact and associate with a guide RNA to cleave single- and double-stranded DNA targets. Here, we report the cryo-electron microscopy structure of Cas12f1 (also known as Cas14a) in complex with a guide RNA and its target DNA. Unexpectedly, the structure revealed that two Cas12f1 molecules assemble with the single guide RNA to recognize the double-stranded DNA target. Each Cas12f1 protomer adopts a different conformation and plays distinct roles in nucleic acid recognition and DNA cleavage, thereby explaining how the miniature Cas12f1 enzyme achieves RNA-guided DNA cleavage as an "asymmetric homodimer." Our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and provide a framework for the development of compact genome-engineering tools critical for therapeutic genome editing.
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Modulation of TCR stimulation and pifithrin-α improve the genomic safety profile of CRISPR-engineered human T cells.
CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.
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Engineering Stable Pseudomonas putida S12 by CRISPR for 2,5-Furandicarboxylic Acid (FDCA) Production.
FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). Pseudomonas putida S12 is promising for FDCA production, but generating stable P. putida S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable P. putida S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO2 and CaCO3 to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating HMFH and HMFT1 (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable P. putida S12 strains for FDCA production.
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Functional validation of CHMP7 as an ADHD risk gene.
Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder of childhood with a strong genetic component. Despite the success of mapping ADHD risk loci, little work has been done to experimentally verify the contribution of these loci to ADHD phenotypes. Meta-analysis of four genome-wide association studies in ADHD suggested CHMP7 as a predisposing gene for ADHD. A DNA variant (rs2294123) mapped to CHMP7 has been shown (via bioinformatic analysis) to have a high likelihood for functionality and correlate with reduced transcript levels. We used CRISPR-Cas9 genome editing to generate a chmp7 zebrafish model for ADHD. chmp7+/- fish showed comparable reductions in mRNA levels to individuals homozygous for the CHMP7 ADHD risk allele. These fish displayed significant hyperactivity over a 24-h period at 6 days post-fertilisation compared to chmp7+/+, but this effect did not persist into juvenile and adulthood stages. In addition, chmp7+/- fish had significantly smaller total brain volumes than chmp7+/+ fish. Finally, the hyperactivity at 6 days post-fertilisation was significantly reduced through the application of methylphenidate, a mainstay pharmacological treatment for ADHD. Overall, this study highlights an important role for CHMP7 in the neurodevelopment of ADHD, and demonstrates the utility of zebrafish for modelling the functional effects of genes conferring risk to ADHD.
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