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Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene.
Achieving stable and efficient transgene expression is a key challenge in advancing avian genome engineering. Although viral vector-based and piggyBac-mediated transgenesis have been widely used in chickens, both approaches are prone to epigenetic silencing, leading to inconsistent, tissue-specific, and often diminished expression over time. This variability limits used of transgenes requiring robust and long-term expression across multiple tissues. In mammals, site-specific integration into genomic safe harbor loci, such as Rosa26, has enabled stable and predictable transgene expression without disrupting endogenous gene function; however, such strategy has not been established in birds. In this research, we hypothesized that integrating Cas9 into endogenous housekeeping genes (the ACTB and GAPDH) could achieve efficient gene editing in chickens through stable and ubiquitous transgene expression. Using two different approaches, 3'-targeted gene insertion and gene tagging, we inserted Cas9 and GFP cassettes into defined genomic loci in chicken DF-1 cells. Both approaches exhibited stable expression of transgenes in the cells, and functional assays confirmed that Cas9 showed highly efficient nuclease activity following guide RNA delivery. Additionally, we derived single-cell clones stably expressing Cas9, enabling uniform and reproducible genome editing in downstream applications. Targeted insertion of transgenes into active housekeeping genes as candidate safe harbor loci mitigates the limitations of random integration and promoter silencing, offering a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.
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Generating adamts13-/- Zebrafish via CRISPR/Cas9 Gene Editing.
The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.
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Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.
The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.
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Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene.
Efficient genetic transformation has the potential to advance research and breeding in watermelon (Citrullus lanatus), but regeneration from tissue culture remains challenging. Previous work showed that expressing a fusion of two interacting transcription factors, GROWTH-REGULATING FACTOR4 (GRF4) and GRF-INTERACTING FACTOR1 (GIF1), improved regeneration in wheat (Triticum aestivum). By overexpressing a chimeric fusion of ClGRF4 and ClGIF1, we achieved highly efficient transformation in watermelon. Mutating the mi396 microRNA target site in ClGRF further boosted the transformation efficiency up to 67.27% in a genotype-independent manner. ClGRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing tools to achieve highly efficient gene editing in watermelon, which we used to successfully create diploid seedless watermelon. This research thus puts forward a powerful transformation tool for future watermelon research and breeding.
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Off-target predictions in CRISPR-Cas9 gene editing using deep learning.
MOTIVATION: The prediction of off-target mutations in CRISPR-Cas9 is a hot topic due to its relevance to gene editing research. Existing prediction methods have been developed; however, most of them just calculated scores based on mismatches to the guide sequence in CRISPR-Cas9. Therefore, the existing prediction methods are unable to scale and improve their performance with the rapid expansion of experimental data in CRISPR-Cas9. Moreover, the existing methods still cannot satisfy enough precision in off-target predictions for gene editing at the clinical level. RESULTS: To address it, we design and implement two algorithms using deep neural networks to predict off-target mutations in CRISPR-Cas9 gene editing (i.e. deep convolutional neural network and deep feedforward neural network). The models were trained and tested on the recently released off-target dataset, CRISPOR dataset, for performance benchmark. Another off-target dataset identified by GUIDE-seq was adopted for additional evaluation. We demonstrate that convolutional neural network achieves the best performance on CRISPOR dataset, yielding an average classification area under the ROC curve (AUC) of 97.2% under stratified 5-fold cross-validation. Interestingly, the deep feedforward neural network can also be competitive at the average AUC of 97.0% under the same setting. We compare the two deep neural network models with the state-of-the-art off-target prediction methods (i.e. CFD, MIT, CROP-IT, and CCTop) and three traditional machine learning models (i.e. random forest, gradient boosting trees, and logistic regression) on both datasets in terms of AUC values, demonstrating the competitive edges of the proposed algorithms. Additional analyses are conducted to investigate the underlying reasons from different perspectives. AVAILABILITY AND IMPLEMENTATION: The example code are available at https://github.com/MichaelLinn/off_target_prediction. The related datasets are available at https://github.com/MichaelLinn/off_target_prediction/tree/master/data.
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Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a.
Considered a serious threat by the Centers for Disease Control and Prevention, multidrug-resistant Enterococcus faecium is an increasing cause of hospital-acquired infection. Here, we provide details on a single-plasmid CRISPR-Cas12a system for generating clean deletions and insertions. Single manipulations were carried out in under 2 weeks, with successful deletions/insertions present in >80% of the clones tested. Using this method, we generated three individual clean deletion mutations in the acpH, treA, and lacL genes and inserted codon-optimized unaG, enabling green fluorescent protein (GFP)-like fluorescence under the control of the trehalase operon. The use of in vivo recombination for plasmid construction kept costs to a minimum. IMPORTANCE Enterococcus faecium is increasingly associated with hard-to-treat antibiotic-resistant infections. The ability to generate clean genomic alterations is the first step in generating a complete mechanistic understanding of how E. faecium acquires pathogenic traits and causes disease. Here, we show that CRISPR-Cas12a can be used to quickly (under 2 weeks) and cheaply delete or insert genes into the E. faecium genome. This substantial improvement over current methods should speed up research on this important opportunistic pathogen.
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Potential of Genome Editing to Improve Aquaculture Breeding and Production.
Aquaculture is the fastest growing food production sector and is rapidly becoming the primary source of seafood for human diets. Selective breeding programs are enabling genetic improvement of production traits, such as disease resistance, but progress is limited by the heritability of the trait and generation interval of the species. New breeding technologies, such as genome editing using CRISPR/Cas9 have the potential to expedite sustainable genetic improvement in aquaculture. Genome editing can rapidly introduce favorable changes to the genome, such as fixing alleles at existing trait loci, creating de novo alleles, or introducing alleles from other strains or species. The high fecundity and external fertilization of most aquaculture species can facilitate genome editing for research and application at a scale that is not possible in farmed terrestrial animals.
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A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing.
The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.
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CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges.
Sickle cell disease (SCD) is an inherited monogenic disorder resulting in serious mortality and morbidity worldwide. Although the disease was characterized more than a century ago, there are only two FDA approved medications to lessen disease severity, and a definitive cure available to all patients with SCD is lacking. Rapid and substantial progress in genome editing approaches have proven valuable as a curative option given plausibility to either correct the underlying mutation in patient-derived hematopoietic stem/progenitor cells (HSPCs), induce fetal hemoglobin expression to circumvent sickling of red blood cells (RBCs), or create corrected induced pluripotent stem cells (iPSCs) among other approaches. Recent discovery of CRISPR/Cas9 has not only revolutionized genome engineering but has also brought the possibility of translating these concepts into a clinically meaningful reality. Here we summarize genome engineering applications using CRISPR/Cas9, addressing challenges and future perspectives of CRISPR/Cas9 as a curative option for SCD.
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A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure.
5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype.
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