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Rewritable multi-event analog recording in bacterial and mammalian cells.
We present two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that use base editors and Cas9 nucleases to record cellular events in bacteria and mammalian cells. The devices record signal amplitude or duration as changes in the ratio of mutually exclusive DNA sequences (CAMERA 1) or as single-base modifications (CAMERA 2). We achieved recording of multiple stimuli in bacteria or mammalian cells, including exposure to antibiotics, nutrients, viruses, light, and changes in Wnt signaling. When recording to multicopy plasmids, reliable readout requires as few as 10 to 100 cells. The order of stimuli can be recorded through an overlapping guide RNA design, and memories can be erased and re-recorded over multiple cycles. CAMERA systems serve as "cell data recorders" that write a history of endogenous or exogenous signaling events into permanent DNA sequence modifications in living cells.
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CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.
RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.
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The Future of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 Gene Therapy in Cardiomyopathies: A Review of Its Therapeutic Potential and Emerging Applications.
Cardiomyopathies, among the leading causes of heart failure and sudden cardiac death, are often driven by genetic mutations affecting the heart's structural proteins. Despite significant advancements in understanding the genetic basis of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), effective long-term therapies remain limited. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing offers a promising therapeutic strategy to address these genetic disorders at their root. CRISPR-Cas9 enables precise modification of pathogenic variants (PVs) in genes encoding sarcomeric and desmosomal proteins, which are frequently implicated in cardiomyopathies. By inducing site-specific double-stranded breaks in DNA, followed by repair through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), this system allows for targeted correction of mutations. In preclinical models, CRISPR-Cas9 has shown promise in correcting HCM-associated mutations in β-myosin heavy chain 7 (MYH7), preventing disease phenotypes such as ventricular hypertrophy and myocardial fibrosis. Similarly, gene editing has successfully rectified DCM-linked mutations in Titin (TTN) and LMNA, resulting in improved heart function and reduced pathological remodeling. For ARVC, CRISPR-Cas9 has demonstrated the ability to repair mutations in desmosomal genes such as plakophilin 2 (PKP2), thereby restoring normal cardiac function and cellular adhesion. Despite these successes, challenges remain, including mosaicism, delivery efficiency, and off-target effects. Nevertheless, CRISPR-Cas9 represents a transformative approach to treating genetic cardiomyopathies, potentially offering long-lasting cures by directly addressing their underlying genetic causes.
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Systematic discovery of natural CRISPR-Cas12a inhibitors.
Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.
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Preventing secondary primary malignancies (SPMs) in CAR-T cell therapy through site-specific transgene integration into genomic safe harbors (GSHs).
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized oncology by achieving durable remissions in refractory hematologic malignancies. However, emerging reports link this therapy to second primary malignancies, including CAR+ lymphomas and leukemias, driven by insertional mutagenesis from semi-random viral vector integration near oncogenes or tumor suppressor loci. These rare but serious complications underscore the dual challenge of eradicating primary tumors while mitigating delayed genotoxic risks. Conventional CAR-T cell manufacturing, reliant on gamma-retroviral or lentiviral vectors, introduces genomic instability through integration into fragile sites or transcriptionally active regions. CRISPR/Cas9-mediated genome editing further amplifies risks via off-target double-strand breaks and chromosomal rearrangements. This review evaluates genomic safe harbors (GSHs)-such as AAVS1, TRAC, CCR5, ROSA26 and CLYBL-as loci validated for stable, high-level CAR transgene expression without oncogenic disruption. GSHs meet stringent criteria: distal from cancer-related genes, resistant to epigenetic silencing, and transcriptionally permissive. Preclinical studies demonstrate that site-directed CAR integration into GSHs preserves antitumor efficacy while eliminating malignant transformation risks. Challenges persist in optimizing homology-directed repair efficiency, mitigating residual dsDNA toxicity, and standardizing regulatory frameworks for long-term genomic surveillance. Emerging technologies-base/prime editing, hybrid nucleases, and rigorous monitoring-promise enhanced precision and safety. By reconciling therapeutic innovation with genomic integrity, GSH-engineered CAR-T cells herald a paradigm shift toward precision immunotherapies, offering curative potential while preempting secondary oncogenesis. Collaborative efforts to refine manufacturing, harmonize global standards, and prioritize patient-specific risk stratification will be critical to advancing this transformative approach.
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Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins), a prokaryotic RNA-mediated adaptive immune system, has been repurposed for gene editing and synthetic gene circuit construction both in bacterial and eukaryotic cells. In the last years, the emergence of the anti-CRISPR proteins (Acrs), which are natural OFF-switches for CRISPR-Cas, has provided a new means to control CRISPR-Cas activity and promoted a further development of CRISPR-Cas-based biotechnological toolkits. In this review, we focus on type I and type V-A anti-CRISPR proteins. We first narrate Acrs discovery and analyze their inhibitory mechanisms from a structural perspective. Then, we describe their applications in gene editing and transcription regulation. Finally, we discuss the potential future usage-and corresponding possible challenges-of these two kinds of anti-CRISPR proteins in eukaryotic synthetic gene circuits.
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Genome edited wheat- current advances for the second green revolution.
Common wheat is a major source of nutrition around the globe, but unlike maize and rice hybrids, no breakthrough has been made to enhance wheat yield since Green Revolution. With the availability of reference genome sequence of wheat and advancement of allied genomics technologies, understanding of genes involved in grain yield components and disease resistance/susceptibility has opened new avenues for crop improvement. Wheat has a huge hexaploidy genome of approximately 17 GB with 85% repetition, and it is a daunting task to induce any mutation across three homeologues that can be helpful for the enhancement of agronomic traits. The CRISPR-Cas9 system provides a promising platform for genome editing in a site-specific manner. In wheat, CRISPR-Cas9 is being used in the improvement of yield, grain quality, biofortification, resistance against diseases, and tolerance against abiotic factors. The promising outcomes of the CRISPR-based multiplexing approach circumvent the constraint of targeting merely one gene at a time. Deployment of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 endonuclease (CRISPR-Cas9) and Cas9 variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce point mutations more precisely. Scientists have acquired major events such as induction of male sterility, fertility restoration, and alteration of seed dormancy through Cas9 in wheat that can facilitate breeding programs for elite variety development. Furthermore, a recent discovery in tissue culturing enables scientists to significantly enhance regeneration efficiency in wheat by transforming the GRF4-GIF1 cassette. Rapid generation advancement by speed breeding technology provides the opportunity for the generation advancement of the desired plants to segregate out unwanted transgenes and allows rapid integration of gene-edited wheat into the breeding pipeline. The combination of these novel technologies addresses some of the most important limiting factors for sustainable and climate-smart wheat that should lead to the second "Green Revolution" for global food security.
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Nucleotide excision repair of abasic DNA lesions.
Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.
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Rapid customization of Solanaceae fruit crops for urban agriculture.
Cultivation of crops in urban environments might reduce the environmental impact of food production1-4. However, lack of available land in cities and a need for rapid crop cycling, to yield quickly and continuously, mean that so far only lettuce and related 'leafy green' vegetables are cultivated in urban farms5. New fruit varieties with architectures and yields suitable for urban farming have proven difficult to breed1,5. We identified a regulator of tomato stem length (SlER) and devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) and precocious growth termination (SP). Application of our strategy using one-step CRISPR-Cas9 genome editing restructured vine-like tomato plants into compact, early yielding plants suitable for urban agriculture. Field data confirmed that yields were maintained, and we demonstrated cultivation in indoor farming systems. Targeting the same stem length regulator alone in groundcherry, another Solanaceae plant, also enabled engineering to a compact stature. Our approach can expand the repertoire of crops for urban agriculture.
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Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.
Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.
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