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At least 19 recordsLinked to original sources
PubMed2026

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.

The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.

Gene Editing↗
PubMed2025

RAPID-DASH: Fast and Efficient Assembly of Guide RNA Arrays for Multiplexed CRISPR-Cas9 Applications.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays. To facilitate the adaptation of this approach, we have made a web tool to design oligo sequences necessary to assemble gRNA arrays.

CRISPR-Cas9↗
PubMed2025

Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

Gene Editing↗
PubMed2025

Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.

Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.

DNA Transposable Elements↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗
PubMed2026

CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.

BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV). METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves. FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82·5% (95% CI 74·0-88·7) for T pallidum and 94·4% (90·2-97·0) for pan-HSV; LODs were 6·2 copies per μL for T pallidum and 7·8 copies per μL for HSV. Reflex testing gave sensitivities of 97·0% (91·1-99·3) for HSV-1 and 96·0% (89·7-98·7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80·0% (74·0-84·9) for N gonorrhoeae and 73·0% (65·5-79·3) for C trachomatis, with a LOD of 3·9 copies per μL for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63·1% (55·1-70·4); however, this was dependent on sample type, with a sensitivity of 85·7% (46·7-99·5) in genital samples and 61·2% (52·8-68·9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0·90 for T pallidum and 0·99 for pan-HSV in the TP-HSV assay, with values of 0·99 for HSV-1 and 0·97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0·90 for N gonorrhoeae and 0·85 for C trachomatis, with a value of 0·72 obtained for gyrA S91F in the reflex cNG-gyrA assay. INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings. FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.

Humans↗
PubMed2026

Multiplexed genome editing by CRISPR-Un1Cas12f1 restores dystrophin expression in a mouse model of Duchenne muscular dystrophy.

The compact type V clustered regularly interspaced short palindromic repeats (CRISPR) nuclease Un1Cas12f1 is compatible with adeno-associated virus (AAV)-mediated genome editing, although the protospacer adjacent motif (PAM) requirements and capacity for multiplexed genome editing remain undefined. Here, we show that Un1Cas12f1 exhibits a broad tolerance for non-canonical PAMs, including Y-rich motifs with a preference for TTCR and TCTA PAMs, thereby expanding the genomic targeting range. We further demonstrate that a tandem sgRNA array expressed from a single transcript supports Un1Cas12f1-mediated multiplexed genome editing at up to five distinct genomic loci. Leveraging this multiplexing capability, we achieved targeted excision of the Dmd exon 23 through intramuscular delivery of an all-in-one AAV vector encoding Un1Cas12f1 and a CRISPR array. This treatment restored the disrupted open reading frame and dystrophin expression in a mouse model of Duchenne muscular dystrophy (DMD). Together, these findings establish Un1Cas12f1 as a compact CRISPR system capable of multiplexed genome editing and demonstrate its therapeutic potential for DMD.

Journal Article↗
PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗
PubMed2025

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗
PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2026

Multiplexed perturbation enables scalable pooled screens.

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

Humans↗
PubMed2026

A rapid CRISPR-based nanodroplet assay enables direct clinical identification of mycobacteria species.

The global incidence and mortality of nontuberculous mycobacterial infections have risen sharply with population aging. In some regions, they are now surpassing Mycobacterium tuberculosis complex infections, imposing a substantial clinical and economic burden. Because nontuberous mycobacteria exhibit species-level heterogeneity and require prolonged culture for identification, their diagnosis remains slow and is frequently inaccurate. Here, we describe a multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-assisted nanodroplet differential identification (CANDI) diagnostic platform that integrates species-agnostic target amplification with species-specific CRISPR-associated protein 12a (Cas12a) detection in fluorescence-barcoded nanodroplets. By spatially compartmentalizing CRISPR reactions into color-encoded nanodroplets, CANDI overcomes the multiplexing limitations of conventional CRISPR diagnostics and enables simultaneous interrogation of multiple mycobacterial targets in a single assay. We designed a 16-plex panel that distinguishes 15 clinically relevant Mycobacterium species and subspecies. CANDI achieved high analytical sensitivity and accurate discrimination in samples containing coinfections with multiple species or subspecies. When applied to 230 clinical specimens, including sputum, tracheal aspirates, and other respiratory fluids, CANDI delivered subspecies-level results within 3.5 hours, achieving 97.08% sensitivity and 99.7% specificity relative to culture-based identification. By combining multiplexed, high-specificity CRISPR detection with scalable droplet-based engineering, CANDI has the potential to overcome the culture dependency of current diagnostics and enable species- and subspecies-level identification across the genetically complex Mycobacterium genus, offering a clinically adaptable framework for rapid, precision diagnosis of mycobacterial infections.

Humans↗
PubMed2025

Endogenous fine-mapping and prioritization of functional regulatory elements in complex genetic loci.

Most genetic loci linked to polygenic traits are in non-coding regions, with complex regulation and linkage disequilibrium (LD), complicating causal variant and gene prioritization. We used multiplexed single-cell CRISPR interference and activation perturbations to investigate cis-regulatory element (CRE) and gene expression relationships within tight LD in the endogenous chromatin context. We demonstrated the prevalence of multiple causality in perfect LD (pLD) for independent expression quantitative trait loci (eQTLs) and uncovered fine-grained genetic effects on gene expression within pLD, which are difficult to decipher using traditional eQTL fine-mapping or existing computational methods. We found that over one-third of the causal CREs lack classical epigenetic markers prior to perturbation, and we functionally validated one of these hidden regulatory mechanisms. Leveraging Multiome single-cell epigenetic and sequence perturbations, we highlighted the regulatory plasticity of the human genome. Our study will guide the exploration of missing causal mechanisms underlying molecular trait regulation and disease development.

Humans↗
PubMed2024

Phase Transition of Wax Enabling CRISPR Diagnostics for Automatic At-Home Testing of Multiple Sexually Transmitted Infection Pathogens.

Sexually transmitted infections (STIs) significantly impact women's reproductive health. Rapid, sensitive, and affordable detection of these pathogens is essential, especially for home-based self-testing, which is crucial for individuals who prioritize privacy or live in areas with limited access to healthcare services. Herein, an automated diagnostic system called Wax-CRISPR has been designed specifically for at-home testing of multiple STIs. This system employs a unique strategy by using the solid-to-liquid phase transition of wax to sequentially isolate and mix recombinase polymerase amplification (RPA) and CRISPR assays in a microfluidic chip. By incorporating a home-built controlling system, Wax-CRISPR achieves true one-pot multiplexed detection. The system can simultaneously detect six common critical gynecological pathogens (CT, MG, UU, NG, HPV 16, and HPV 18) within 30 min, with a detection limit reaching 10-18 M. Clinical evaluation demonstrates that the system achieves a sensitivity of 96.8% and a specificity of 97.3% across 100 clinical samples. Importantly, eight randomly recruited untrained operators performe a double-blinded test and successfully identified the STI targets in 33 clinical samples. This wax-transition-based one-pot CRISPR assay offers advantages such as low-cost, high-stability, and user-friendliness, making it a useful platform for at-home or field-based testing of multiple pathogen infections.

Sexually Transmitted Diseases↗
PubMed2026

CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.

Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.

CRISPR↗
PubMed2026

CRISPR-Cas technologies for precision genome editing in plants: advances, applications, and future perspectives.

Developing climate-smart crops with enhanced crop productivity, nutritional quality, resistance to biological and environmental stressors is vital for global food security. While hybrid breeding forms the cornerstone of modern crop improvement, conventional breeding approaches are limited by genetic barriers and prolonged breeding cycles. CRISPR-Cas based genome editing has revolutionized plant biology by allowing precise, efficient, and multiplex genetic modifications. This review provides a comprehensive synthesis of a recent advances in CRISPR-Cas technologies and their strategic applications in crop genetics and hybrid breeding. We summarize major genome-editing strategies, including gene knock-out, base editing (BE), knock-in, gene replacement, epigenome editing, and transcriptional regulation. Furthermore, we contrast stable, transient, and DNA-free delivery systems, highlighting ribonucleoprotein (RNP)-mediated delivery for minimizing off-target effects and avoiding transgene integration. We showcase how these technologies accelerate hybrid breeding by engineering male sterility systems, fixing heterosis, and generating high-throughput mutant libraries for trait discovery. Finally, we synthesize major bottlenecks in tissue culture-independent transformation and delivery systems, while outlining how emerging paradigms like de novo domestication and synthetic biology will shape the future of climate-resilient agriculture.

CRISPR/Cas↗
PubMed2025

HyperCas12a enables multiplexed CRISPRi screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We further show that the system can be used in cultured primary immune cells and to drive differentiation of induced pluripotent stem cells. We also developed new approaches to use the dCas12a platform for simultaneous activation and repression from a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, we demonstrate that the dHyperLbCas12a effectors are highly effective for multiple modalities of high-throughput screens, namely proliferation screens and screens to dissect the independent and combinatorial contributions of CREs on gene expression. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

Journal Article↗