Genome Editing Research Articles

CRISPR in clinical diagnostics: bridging the gap between research and practice.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has transformed molecular biology through its precise gene-editing capabilities. Beyond its initial applications in genetic modification, CRISPR has emerged as a powerful tool in diagnostics and biosensing. This review explores its transition from genome editing to innovative detection methods, including nucleic acid identification, single nucleotide polymorphism (SNP) analysis, and protein sensing. Advanced technologies such as SHERLOCK and DETECTR demonstrate CRISPR's potential for point-of-care diagnostics, enabling rapid and highly sensitive detection. The integration of chemical modifications, CRISPR-Chip technology, and enzymatic systems like Cas12a and Cas13a enhances signal amplification and detection efficiency. These advancements promise decentralized, real-time diagnostic solutions with significant implications for global healthcare. Furthermore, the fusion of CRISPR with artificial intelligence and digital health platforms is paving the way for more accessible, cost-effective, and scalable diagnostic approaches, ultimately revolutionizing precision medicine.

Enhancing lipid production in plant cells through automated high-throughput genome editing and phenotyping

Abstract Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyl fluorescence 136 (HCF136). We show that diverse lipids were enhanced up to sixfold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multi-gene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

Advancements in genome editing tools for genetic studies and crop improvement

The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.

Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing.

The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies has opened up groundbreaking possibilities for treating a wide spectrum of genetic disorders and diseases. However, the success of these technologies relies heavily on the development of efficient and safe delivery systems. Among the most promising approaches are natural and synthetic nanocarrier-mediated delivery systems, including viral vectors, extracellular vesicles (EVs), engineered cellular membrane particles, liposomes, and various nanoparticles. These carriers enhance the efficacy of the CRISPR system by providing a unique combination of efficiency, specificity, and reduced immunogenicity. Synthetic carriers such as liposomes and nanoparticles facilitate CRISPR delivery with high reproducibility and customizable functions. Viral vectors, renowned for their high transduction efficiency and broad tropism, serve as powerful vehicles for delivering CRISPR components to various cell types. EVs, as natural carriers of RNA and proteins, offer a stealth mechanism to evade immune detection, allowing for the targeted delivery of genome editors with minimal off-target effects. Engineered cellular membrane particles further improve delivery by simulating the cellular environment, enhancing uptake, and minimizing immune response. This review explores the innovative integration of CRISPR genome editors with various nanocarrier systems, focusing on recent advancements, applications, and future directions in therapeutic genome editing.

Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells

Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.

Gene Editing for Enhanced Swine Production: Current Advances and Prospects

Traditional pig breeding has improved production traits but faces limitations in genetic diversity, disease resistance, and environmental adaptation. Gene editing technologies, such as CRISPR/Cas9, base editing, and prime editing, enable precise genetic modifications, overcoming these limitations and expanding applications to biomedical research. Here, we reviewed the advancements in gene editing technologies in pigs and explored pathways toward optimized swine genetics for a resilient and adaptive livestock industry. This review synthesizes recent research on gene editing tools applied to pigs, focusing on CRISPR/Cas9 and its derivatives. It examines their impact on critical swine production traits and their role as human disease models. Significant advancements have been made in targeting genes for disease resistance, such as those conferring immunity to porcine reproductive and respiratory syndrome viruses. Additionally, gene-edited pigs are increasingly used as models for human diseases, demonstrating the technology’s broader applications. However, challenges such as off-target effects, ethical concerns, and varying regulatory frameworks remain. Gene editing holds substantial potential for sustainable and productive livestock production by enhancing key traits and supporting biomedical applications. Addressing technical and ethical challenges through integrated approaches will be essential to realize its full potential, ensuring a resilient, ethical, and productive livestock sector for future generations

Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders.

The high frequency of genetic diseases compels the development of refined diagnostic and therapeutic systems. CRISPR is a precise genome editing tool that offers detection of genetic mutation with high sensitivity, specificity and flexibility for point-of-care testing in low resource environment. Advancements in CRISPR ushered new hope for the detection of genetic diseases. This review aims to explore the recent advances in CRISPR for the detection and treatment of genetic disorders. It delves into the advances like next-generation CRISPR diagnostics like nano-biosensors, digitalized CRISPR, and omics-integrated CRISPR technologies to enhance the detection limits and to facilitate the "lab-on-chip" technologies. Additionally, therapeutic potential of CRISPR technologies is reviewed to evaluate the implementation potential of CRISPR technologies for the treatment of hematological diseases, (sickle cell anemia and β-thalassemia), HIV, cancer, cardiovascular diseases, and neurological disorders, etc. Emerging CRISPR therapeutic approaches such as base/epigenetic editing and stem cells for the development of foreseen CRIPSR drugs are explored for the development of point-of-care testing. A combination of predictive models of artificial intelligence and machine learning with growing knowledge of genetic disorders has also been discussed to understand their role in acceleration of genetic detection. Ethical consideration are briefly discussed towards to end of review. This review provides the comprehensive insights into advances in the CRISPR diagnostics/therapeutics which are believed to pave the way for reliable, effective, and low-cost genetic testing.

Effects of Electroporation Timing and Cumulus Cell Attachment on InVitro Development and Genome Editing of Porcine Embryos.

Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before invitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.

MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering.

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.

CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.

Valuation in translation of genomic medicine technologies

Valuation in translation of genomic medicine technologies

Immune regulation differences in Large yellow croaker with varied resistance to Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the main marine aquaculture species in China, but has faced considerable losses due to Cryptocaryon irritans infection. In this study, we successfully established a C. irritans-susceptible population of large yellow croaker by genomic selection technology. We then compared the immune genetic mechanisms of this susceptible population with those of a large yellow croaker population from eastern Fujian in response to C. irritans infection. GWAS identified 44 significant SNPs across 11 QTL regions on different chromosomes associated with C. irritans infection, with most located on chromosomes 1 and 24. Notably, the QTL region on chromosome 1 overlapped with the resistance QTL region mapped in the C. irritans-resistant population previously established by our team, underscoring its crucial role in conferring resistance to C. irritans infection. RNA-Seq analysis revealed significant differences in immune responses between the two groups, with the susceptible group specifically activating the Jak / Stat signaling pathway and upregulating interleukin-related genes, including il11a, il-5r and il-20r. A combined analysis of the GWAS and RNA-Seq data revealed that cspg4 was located in the overlapping QTL region on chromosome 1 associated with resistance. Upon infection, the expression of cspg4 was significantly higher in the susceptible group compared to the control group. As a downstream factor of interleukins, cspg4 may regulate interleukin expression by activating the Jak / Stat pathway, thereby influencing the body's normal immune defense functions. These findings provide new insights into the mechanisms of host-parasite immune responses and highlight potential therapeutic targets.

Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.

Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.

Advances in lignocellulosic feedstocks for bioenergy and bioproducts.

Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.

Using omics data and genome editing methods to decipher GWAS loci associated with coronary artery disease

Using omics data and genome editing methods to decipher GWAS loci associated with coronary artery disease

Unlocking the potential of CMP synthesis: Merging enzyme design, host genome editing, and fermentation optimization strategy

Unlocking the potential of CMP synthesis: Merging enzyme design, host genome editing, and fermentation optimization strategy

A CRISPR view on genetic screens inToxoplasma gondii.

Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety of organisms, including unicellular parasites. Today, the CRISPR-Cas9 technology is vastly applied in high-throughput screens to investigate interactions between the Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro and in vivo T. gondii screens performed in naive and restrictive conditions have led to the discovery of essential and fitness-conferring T. gondii genes, as well as factors important for virulence and dissemination. Recent studies have adapted the CRISPR-Cas9 screening technology to study T. gondii genes based on phenotypes unrelated to parasite survival. These advances were achieved by using conditional systems coupled with imaging, as well as single-cell RNA sequencing and phenotypic selection. Here, we review the state-of-the-art of CRISPR-Cas9 screening technologies with a focus on T. gondii, highlighting strengths, current limitations and future avenues for its development, including its application to other Apicomplexan species.

Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.

The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.

Understanding Heritable Variation Among Hosts in Infectious Diseases Through the Lens of Twin Studies

Genetic factors have been hypothesized to contribute to the heterogeneity in the response to infectious diseases (IDs). The classical twin design provides a powerful tool to estimate the role of genetic contributions to variation in infection outcomes. With this design, the impact of heritability on the proneness as well as infection- and vaccine-induced immune responses have been documented for multiple infections, including tuberculosis, malaria, leprosy, otitis media, polio, mumps, measles, rubella, influenza, hepatitis B, and human papillomavirus infections, and recently, SARS-CoV-2. The current data show the heritable aspect in nearly all infections considered. In this contribution, we review and discuss human twin studies on the heritability of host characteristics in liability and response to IDs. This review emphasizes the importance of considering factors such as sex, disease stages, and disease presentation when assessing heritability and argues that the classical twin design provides a unique circumstance for exploring the genetic contribution as twins share levels of maternal antibodies, ancestral background, often the dates and number of vaccine doses, differences in vaccines’ manufacturing and storage, age, family environment, and other exposures. Additionally, we highlight the value of twin studies and the usefulness of combining the twin model with contemporary genomics technologies and advanced statistical tools to grasp a comprehensive and nuanced understanding of heritability in IDs.

CRISPR-guided base editor enables efficient and multiplex genome editing in bacterial cellulose-producing Komagataeibacter species.

Komagataeibacter, a bacterial genus belonging to the family Acetobacteraceae, has important applications in food and material biosynthesis. However, the genome editing of Komagataeibacter relies on traditional homologous recombination methods. Therefore, only one gene can be manipulated in each round using foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms when microorganisms are used in the food industry. In this study, a powerful base editing technology was developed for Komagataeibacter species. C-to-T and C-to-G base conversions were efficiently implemented at up to three loci in the Komagataeibacter genome. This base editing system is expected to accelerate basic and applied research on Komagataeibacter species.