Targeted large-fragment genomic deletion in human pluripotent stem cells (hPSCs) via CRISPR/Cas9.

Enhancing CRISPR/Cas-mediated detection of nucleic acids using PNIPAM-based reporters.

A cleavage-gated terminal exposure-driven CRISPR-RCA self-amplifying system for ultra-fast DNA detection.

Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.

The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3′-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.

Proper neural circuit organization requires individual neurons to project to their targets with high specificity. While several guidance molecules have been shown to mediate axonal fasciculation and pathfinding, less is understood about how neurons intracellularly interpret and integrate these cues. Here we provide genetic evidence that the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins is required for proper fasciculation and guidance of two cortical white matter tracts: the Anterior Commissure (AC) and thalamocortical axons (TCAs). Using a Cas Triple Conditional Knock Out (Cas TcKO) mouse model, we show that Cas proteins are required for proper TCA projection by a non-neuronal cortical cell population. We also demonstrate a requirement of the β1-integrin receptor for TCA projection, similarly in a population of non-neuronal cortical cells. Additional analysis of Cas TcKO mutants reveals a role for Cas proteins in AC fasciculation, here within the neurons themselves. This AC fasciculation requirement is not phenocopied in β1-integrin deficient mutants, suggesting that Cas proteins might signal downstream of a different receptor during this axon pathfinding event. These findings implicate Cas proteins as key mediators of cortical axon tract fasciculation and guidance.

CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.

Patient-informed CRISPR screening reveals gene targets to improve CAR T-cell efficacy

Single-step purification of functional Cas9 protein via the ubiquitin expression system.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR–Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39-71% of transformed calli by PCR analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice; base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.

Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant–virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host–virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.

CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools. Methods: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST. Results: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems. Conclusions: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.

The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.

Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.

The stability of CRISPR-Cas9 endonuclease activity is essential for its effectiveness in molecular diagnostics and gene editing. Target sequences of Human Papillomavirus (HPV) types 16 and 18 were selected by generating a consensus sequence following multiple sequence alignment, to ensure high specificity. The single guide RNAs (sgRNAs) were designed by identifying Protospacer Adjacent Motif (PAM) sequences within the E6 gene of HPV-16 and HPV-18. High-fidelity DNA oligonucleotides were synthesized from Integrated DNA Technologies (IDT) and transcribed in vitro into single guide RNAs (sgRNAs). These sgRNAs were then assembled with Cas9 protein to form CRISPR-Cas9 ribonucleoprotein (RNP) complexes, which were subsequently lyophilized to enhance storage stability. Functional validation of the RNP complexes was performed over a period of up to 18 months using polymerase chain reaction (PCR), agarose gel electrophoresis (AGE), and SYBR Green-based real-time PCR (RT-PCR) to confirm endonuclease activity and cleavage efficiency. This study assessed the activity of lyophilized HPV-16 and HPV-18 CRISPR-Cas9-RNPs stored at 4 °C for up to 18 months. Results demonstrated that the ribonucleoprotein (RNP) complex consisting sgRNA retained significant endonuclease activity, supporting lyophilization as a viable strategy for enhancing the stability and shelf life of CRISPR-Cas9 complexes for long-term applications.

A reversible genetic NOR gate in plants using translational repression.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive polyphagous pest that causes significant damage to horticultural crops of global importance. Traditional management practices have not been effective in controlling this pest, and therefore, there is a need for alternative management strategies. CRISPR/Cas9-driven genome editing has been successfully used in a wide range of insects to induce site-specific, off-target minimized mutations that result in loss of function. This technique can be used to develop precision-guided sterile insect technique (pgSIT) and gene drive programs, which can be used for area-wide suppression of the pest. This chapter provides a brief overview of the workflow for RNP-based genome editing, which can be used to validate and establish gene function for large-scale gene drive programs aimed at combating this pest. The RNP, or ribonucleoprotein complex, comprises the sgRNA and Cas9 protein, which are microinjected into the G0 stage embryos for heritable editing of the target gene(s).

Expanding horizons of CRISPR applications beyond genome editing.