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Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a key role in somatic hypermutation and class switch recombination in germinal center B cells. However, off-target effects of AID are implicated in human leukemia and lymphoma. A mouse model of precursor B cell acute lymphoblastic leukemia driven by deletion of the related transcription factors PU.1 and Spi-B revealed C->T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Aicda mRNA transcript levels were increased in leukemia cells and bone marrow pre-B cells lacking PU.1 and/or Spi-B, relative to wild type cells. Using chromatin immunoprecipitation, PU.1 was found to interact with a negative regulatory region (R2–1) within the first intron of Aicda. CRISPR-Cas9-induced mutagenesis of R2–1 in cultured pre-B cells resulted in upregulation of Aicda in response to lipopolysaccharide stimulation. Mutation of the PU.1 interaction site and neighboring sequences resulted in reduced repressive ability of R2–1 in transient transfection analysis followed by luciferase assays. These results show that a PU.1-interacting intronic region negatively regulates Aicda transcription in developing B cells. • Aicda transcription is dysregulated in the absence of PU.1. • The transcription factor PU.1 interacts with the first intron of Aicda. • CRISPR-Cas9 mutation of a PU.1 site in Aicda intron 1 dysregulates transcription. • A PU.1-interacting site in intron 1 is a negative regulator of Aicda transcription. [ABSTRACT FROM AUTHOR]

• Cre recombinase targeted to noradrenergic neurons via CRISPR-Cas9. • High specificity of Cre expression validated in locus coeruleus. • New transgenic rat model facilitates detailed neurobehavioral studies. Noradrenergic neurons play a crucial role in the functioning of the nervous system. They formed compact small clusters in the central nervous system. To target noradrenergic neurons in combination with viral tracing and achieve cell-type specific functional manipulation using chemogenetic or optogenetic tools, new transgenic animal lines are needed, especially rat models for their advantages in large body size with facilitating easy operation, physiological parameter monitoring, and accommodating complex behavioral and cognitive studies. In this study, we successfully generated a transgenic rat strain capable of expressing Cre recombinase under the control of the dopamine beta-hydroxylase (DBH) gene promoter using the CRISPR-Cas9 system. Our validation process included co-immunostaining with Cre and DBH antibodies, confirming the specific expression of Cre recombinase. Furthermore, stereotaxic injection of a fluorescence-labeled AAV-DIO virus illustrated the precise Cre-loxP-mediated recombination activity in noradrenergic neurons within the locus coeruleus (LC). Through crossbreeding with the LSL-fluorescence reporter rat line, DBH-Cre rats proved instrumental in delineating the position and structure of noradrenergic neuron clusters A1, A2, A6 (LC), and A7 in rats. Additionally, our specific activation of the LC noradrenergic neurons showed effective behavioral readout using chemogenetics of this rat line. Our results underscore the effectiveness and specificity of Cre recombinase in noradrenergic neurons, serving as a robust tool for cell-type specific targeting of small-sized noradrenergic nuclei. This approach enhances our understanding of their anatomical, physiological, and pathological roles, contributing to a more profound comprehension of noradrenergic neuron function in the nervous system. [ABSTRACT FROM AUTHOR]

The predominant genetic defense mechanism against soybean cyst nematode (SCN) in 95% of the North America market is under threat by virulent SCN populations. Usovsky et al. identified GmSNAP02 as an SCN susceptibility gene through fine-mapping of unique bi-parental populations. Loss-of-function of GmSNAP02 confers enhanced resistance to more virulent SCN. [ABSTRACT FROM AUTHOR]

Copper-based nanomaterials demonstrate promising potential in cancer therapy. Cu+ efficiently triggers a Fenton-like reaction and further consumes the high level of glutathione, initiating chemical dynamic therapy (CDT) and ferroptosis. Cuproptosis, a newly identified cell death modality that represents a great prospect in cancer therapy, is activated. However, active homeostatic systems rigorously keep copper levels within cells exceptionally low, which hinders the application of cooper nanomaterials-based therapy. Herein, a novel strategy of CRISPR-Cas9 RNP nanocarrier to deliver cuprous ions and suppress the expression of copper transporter protein ATP7A for maintaining a high level of copper in cytoplasmic fluid is developed. The Cu 2 O and organosilica shell would degrade under the high level of glutathione and weak acidic environment, further releasing RNP and Cu+. The liberated Cu+ triggered a Fenton-like reaction for CDT and partially transformed to Cu2+, consuming intracellular GSH and initiating cuproptosis and ferroptosis efficiently. Meanwhile, the release of RNP effectively reduced the expression of copper transporter ATP7A, subsequently increasing the accumulation of cooper and enhancing the efficacy of CDT, cuproptosis, and ferroptosis. Such tumor microenvironment responsive multimodal nanoplatform opens an ingenious avenue for colorectal cancer therapy based on gene editing enhanced synergistic cuproptosis/CDT/ferroptosis. A Cu 2 O-based, CRISPR-Cas9 RNP-doped, organosilica shell-coated and folic acid modified polymorphous therapeutic nanomedicine (RNP@Cu 2 O@SPF) was prepared to convey RNP and Cu+ into tumor cells for gene editing promoted synergistic cuproptosis/chemodynamic/ferroptosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT -edited heterozygotes (KIT D17/+ ). The KIT D17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour. • Cloned cats with KIT edited by CRISPR-Cas9 presented a distinct coat colour variation. • Western blotting showed impaired proliferation and differentiation of melanoblasts in back skin of clone cats. • The findings provide a basis for the breeding of cats with commercially desired coat colour. [ABSTRACT FROM AUTHOR]

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A -edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease. [ABSTRACT FROM AUTHOR]

The emergence of gene editing technologies offers a unique opportunity to develop mutation-specific treatments for pediatric neurological disorders. Gene editing systems can potentially alter disease trajectory by correcting dysfunctional mutations or therapeutically altering gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches are attractive gene therapy platforms to personalize treatments because of their specificity, ease of design, versatility, and cost. However, many such approaches remain in the early stages of development, with ongoing efforts to optimize editing efficiency, minimize unintended off-target effects, and mitigate pathologic immune responses. Given the rapid evolution of CRISPR-based therapies, it is prudent for the clinically based child neurologist to have a conceptual understanding of what such therapies may entail, including both benefits and risks and how such therapies may be clinically applied. In this review, we describe the fundamentals of CRISPR-based therapies, discuss the opportunities and challenges that have arisen, and highlight preclinical work in several pediatric neurological diseases. [ABSTRACT FROM AUTHOR]

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases, immune cell infiltration and protease-driven tissue damages. It is an urgent need to explore potential drug strategies for mitigating lung inflammation. Protease-activated receptor 2 (PAR2) as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction. However, it has been rarely reported about the role of PAR2 in lung inflammation. This study applied CRISPR-Cas9 system encoding Cas9 and sgRNA (pCas9-PAR2) for PAR2 knockout and fabricated an anionic human serum albumin-based nanoparticles to deliver pCas9-PAR2 with superior inflammation-targeting efficiency and stability (TAP /pCas9-PAR2). TAP /pCas9-PAR2 robustly facilitated pCas9-PAR2 to enter and transfect inflammatory cells, eliciting precise gene editing of PAR2 in vitro and in vivo. Importantly, PAR2 deficiency by TAP /pCas9-PAR2 effectively and safely promoted macrophage polarization, suppressed pro-inflammatory cytokine releases and alleviated acute lung inflammation, uncovering a novel value of PAR2. It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP /pCas9-PAR2 was mainly dependent on ERK-mediated NLRP3/IL-1 β and NO/iNOS signalling. Therefore, this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases. TAP/ pCas9-PAR2 alleviated acute lung inflammation through ERK/NLRP3/IL-1 β and NO/iNOS signalling via genome editing of PAR2. [Display omitted] [ABSTRACT FROM AUTHOR]

Cancer is the second leading cause of death worldwide, despite recent advances in its identification and management. To improve cancer patient diagnosis and care, it is necessary to identify new biomarkers and molecular targets. In recent years, long non-coding RNAs (lncRNAs) have surfaced as important contributors to various cellular activities, with growing proof indicating their substantial role in the genesis, development, and spread of cancer. Their unique expression profiles within specific tissues and their wide-ranging functionalities make lncRNAs excellent candidates for potential therapeutic intervention in cancer management. They are implicated in multiple hallmarks of cancer, such as uncontrolled proliferation, angiogenesis, and immune evasion. This review article explores the innovative application of CRISPR-Cas9 technology in targeting lncRNAs as a cancer therapeutic strategy. The CRISPR-Cas9 system has been widely applied in functional genomics, gene therapy, and cancer research, offering a versatile platform for lncRNA targeting. CRISPR-Cas9-mediated targeting of lncRNAs can be achieved through CRISPR interference, activation or the complete knockout of lncRNA loci. Combining CRISPR-Cas9 technology with high-throughput functional genomics makes it possible to identify lncRNAs critical for the survival of specific cancer subtypes, opening the door for tailored treatments and personalised cancer therapies. CRISPR-Cas9-mediated lncRNA targeting with other cutting-edge cancer therapies, such as immunotherapy and targeted molecular therapeutics can be used to overcome the drug resistance in cancer. The synergy of lncRNA research and CRISPR-Cas9 technology presents immense potential for individualized cancer treatment, offering renewed hope in the battle against this disease. • LncRNAs contribute to cellular activities and play a role in genesis, development, and spread of cancer. • Their unique expression profiles within specific tissues make them excellent candidates for cancer therapy. • CRISPR-Cas9 system is versatile and can target lncRNAs. • CRISPR-Cas9 technology with high-throughput functional genomics can identify lncRNAs critical for specific cancer subtypes. [ABSTRACT FROM AUTHOR]

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus , repair of CRISPR-induced double strand breaks is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeSc CHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development. • CRISPR editing in allo-aneuploid yeasts results mostly in loss of heterozygosity. • In S. pastorianus CBS1483, SeSc CHRIII has no homoeologous chromosome. • Genomic locations devoid of homoeologous counterpart are proper integrations sites. • S. pastorianus strains CNV analysis reveals pervasive presence of chimeric SeSc CHRIII. • Genome editing of lager yeast will accelerate future strain development. [ABSTRACT FROM AUTHOR]

Bacterial populations exhibit heterogeneity in gene expression, which facilitates their survival and adaptation to unstable and unpredictable environments through the bet-hedging strategy. However, unraveling the rare subpopulations and heterogeneity in gene expression using population-level gene expression analysis remains a challenging task. Single-cell RNA sequencing (scRNA-seq) has the potential to identify rare subpopulations and capture heterogeneity in bacterial populations, but standard methods for scRNA-seq in bacteria are still under development, mainly due to differences in mRNA abundance and structure between eukaryotic and prokaryotic organisms. In this study, we present a hybrid approach that combines random displacement amplification sequencing (RamDA-seq) with Cas9-based rRNA depletion for scRNA-seq in bacteria. This approach allows cDNA amplification and subsequent sequencing library preparation from low-abundance bacterial RNAs. We evaluated its sequenced read proportion, gene detection sensitivity, and gene expression patterns from the dilution series of total RNA or the sorted single Escherichia coli cells. Our results demonstrated the detection of more than 1000 genes, about 24% of the genes in the E. coli genome, from single cells with less sequencing effort compared to conventional methods. We observed gene expression clusters between different cellular proliferation states or heat shock treatment. The approach demonstrated high detection sensitivity in gene expression analysis compared to current bacterial scRNA-seq methods and proved to be an invaluable tool for understanding the ecology of bacterial populations and capturing the heterogeneity of bacterial gene expression. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are genotoxic lesions that induce extensive chromatin modifications and 3D genome reorganization. Comprehensive interrogation of DSB repair is incomplete, particularly at high spatiotemporal resolution and in the context of various sequences and diverse chromatin environments. Emerging CRISPR technologies rapidly induce highly synchronized DSBs at predetermined targets, enabling kinetic analyses of DSB repair with exquisite temporal control. The marriage of CRISPR-based DSB induction and genome imaging allows DSB repair dynamics to be visualized at specific loci and within single cells. DNA double-strand breaks (DSBs) are one of the most genotoxic DNA lesions, driving a range of pathological defects from cancers to immunodeficiencies. To combat genomic instability caused by DSBs, evolution has outfitted cells with an intricate protein network dedicated to the rapid and accurate repair of these lesions. Pioneering studies have identified and characterized many crucial repair factors in this network, while the advent of genome manipulation tools like clustered regularly interspersed short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) has reinvigorated interest in DSB repair mechanisms. This review surveys the latest methodological advances and biological insights gained by utilizing Cas9 as a precise 'damage inducer' for the study of DSB repair. We highlight rapidly inducible Cas9 systems that enable synchronized and efficient break induction. When combined with sequencing and genome-specific imaging approaches, inducible Cas9 systems greatly expand our capability to spatiotemporally characterize cellular responses to DSB at specific genomic coordinates, providing mechanistic insights that were previously unobtainable. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique. Adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging requires consideration of (amplified) signal generation complexity, an appropriate cell delivery mechanism, and the capacity for single-copy sequence imaging. Single-copy sequence imaging is achieved both with and without signal amplification, whereas SNP or single-nucleotide variant (SNV) detection requires signal amplification. CRISPR-based imaging with specificity at the SNV and SNP levels was achieved in both live and fixed cells. The versatility of paired RNA aptamer-based signal generation enables highly multiplexed live-cell imaging of six repetitive sequences. CRISPR-based imaging was used in a clinical setting, allowing the detection of Patau syndrome (in the form of Chr13 trisomy) in patient-derived cells. [ABSTRACT FROM AUTHOR]

Genetic drive represents a fundamental force of evolution that operates by biasing allele transmission by individuals in a population. Genetic engineering now makes the anthropogenic manipulation of genetic drive feasible in a process termed 'genetic welding'. Genetic welding with synthetic gene drives, which is similar to but distinct from artificial selection, is extremely powerful and profoundly capable of promoting the evolution of complex and novel phenotypic changes to both captive and wild populations. Bioethical analysis and biological research into genetic welding of natural populations with synthetic gene drives requires further consideration, including potential application to humans and the long-term evolutionary consequences of synthetic gene drive elements. Genetic drive represents a fundamental evolutionary force that can exact profound change to the genetic composition of populations by biasing allele transmission. Herein I propose that the use of synthetic homing gene drives, the human-mediated analog of endogenous genetic drives, warrants the designation of 'genetic welding' as an anthropogenic evolutionary force. Conceptually, this distinction parallels that of artificial and natural selection. Genetic welding is capable of imposing complex and rapid heritable phenotypic change on entire populations, whether motivated by biodiversity conservation or public health. Unanticipated possible long-term evolutionary outcomes, however, demand further investigation and bioethical consideration. The emerging importance of genetic welding also compels our explicit recognition of genetic drive as an addition to the other four fundamental forces of evolution. [ABSTRACT FROM AUTHOR]

Genome editing of hematopoietic stem and progenitor cells is being developed for the treatment of several inherited disorders of the hematopoietic system. The adaptation of CRISPR-Cas9-based technologies to make precise changes to the genome , and developments in altering the specificity and efficiency, and improving the delivery of nucleases to target cells have led to several breakthroughs. Many clinical trials are ongoing, and several pre-clinical models have been reported that would allow these genetic therapies to one day offer a potential cure to patients with diseases where limited options currently exist. However, there remain several challenges with respect to establishing safety, expanding accessibility and improving the manufacturing processes of these therapeutic products. This review focuses on some of the recent advances in the field of genome editing of hematopoietic stem and progenitor cells and illustrates the ongoing challenges. [ABSTRACT FROM AUTHOR]

[Display omitted] • The physiological role of the phosphatase activity of the bifunctional sEH is unknown. • CRISPR/Cas9-mediated inhibition of sEH phosphatase in rats induces a lean phenotype. • Potentiation of brown adipose tissue thermogenesis drives increased energy expenditure in sEH phosphatase knock-in rats. • sEH phosphatase knock-in rats have an increased cardiac contractility and were protected against cardiac ischemia–reperfusion injury. • The phosphatase activity of sEH represents a new potential target to fight against obesity and cardiac ischemic complications. Although the physiological role of the C -terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N -terminal phosphatase activity (sEH-P) remains unknown. This study aimed to assess in vivo the physiological role of sEH-P. CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia–reperfusion injury. Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications. [ABSTRACT FROM AUTHOR]

Gene editing technologies, particularly CRISPR-Cas9, have revolutionized agriculture by offering precise and efficient tools to enhance crop production. The vegetatively propagated crops, crucial for global food security, face challenges such as climate change, pests, and limited genetic diversity. CRISPR-Cas9 enables targeted modifications to improve traits like disease resistance, drought tolerance, and nutritional content, thereby boosting productivity and sustainability. Despite its transformative potential, the adoption of gene editing in vegetatively propagated crops is hampered by technical complexities and regulatory frameworks. This review explores recent advancements, challenges, and prospects of gene editing in vegetatively propagated crops, emphasizing strategies to overcome technical barriers and regulatory constraints. Addressing these issues is essential for realizing the full agricultural potential of gene editing and ensuring food security in a changing global climate. [ABSTRACT FROM AUTHOR]

S-adenosyl-L-methionine (SAM) plays pivotal roles in various physiological processes. With its increasing application in the treatment of diseases such as liver disease, depression, osteoarthritis and Alzheimer's, interest in SAM production aroused. Currently, Saccharomyces cerevisiae is the main industrial producer of SAM. With the surge in demand for SAM, improving the SAM biosynthesis is of importance. In this study, a multimodule engineering strategy was employed to improving SAM production: 1) Enhancing the gene expression of the sulfur assimilation pathway; 2) Strengthening the metabolic flux of the SAM synthesis pathway; 3) Weakening the SAM degradation pathway; 4) Increasing ATP supply. The resulting engineered mutant SC06 (S. cerevisiae CEN.PK2–1CΔ gal 80 ∷T cyc1 - sam2-P gal1 -P gal10 -met14-T adh1 , Δ lsc2∷T cyc1 - hom6-P gal1 -P gal10 -met6-T adh1 , Δ sah1 Δ mls1) displayed the highest SAM titer of 240.86 mg/L, which was 10.22-fold increase compared with the original strain. With optimized conditions, the SAM titer of mutant SC06 in shake flask fermentation reached 473.02 mg/L with a specific yield of 127.18 mg/g dry cell weight (DCW). In a 5 L fermenter with fed-batch fermentation, the maximal SAM yield of mutant SC06 reached 1.25 g/L with a specific yield of 166.67 mg/g DCW after 58 h cultivation. Therefore, the established metabolic engineering strategies displayed promising efficiency in improving the SAM productivity of S. cerevisiae CEN.PK2–1C, which may provide a useful tool for the improvement of SAM-producing strains. [Display omitted] • Overexpressing met14 gene of the sulfur assimilation pathway elevates SAM synthesis. • Disrupting TCA cycle enhances metabolic flux into the SAM synthesis pathway. • Knockout of sah1 gene inhibits SAM consumption and benefits SAM accumulation. • Knockout of mls 1 gene elevates acetyl-CoA level and enhances ATP supply for SAM. • SAM biosynthesis markedly elevated with the metabolic engineering strategies. [ABSTRACT FROM AUTHOR]

[Display omitted] • CrHPR1 was first confirmed as a pivotal target for efficient glycolate biosynthesis. • Mixotrophic conditions demonstrate great capacity to boost glycolate biosynthesis. • The deficiency of cell wall facilitates excretion of glycolate into medium. • Both cell density and glycolate yield were synchronously enhanced. • A new microalgal platform was developed for efficient glycolate production. Microalgae-based glycolate production through the photorespiratory pathway is considered an environmentally friendly approach. However, the potential for glycolate production is limited by photoautotrophic cultivation with low cell density and existing strains. In this study, a targeted knockout approach was used to disrupt the key photorespiration enzyme, Chlamydomonas reinhardtii hydroxypyruvate reductase 1 (CrHPR1), leading to a significant increase in glycolate production of 280.1 mg/L/OD 750. The highest potency yield reached 2.1 g/L under optimized mixotrophic conditions, demonstrating the possibility of synchronizing cell growth with glycolate biosynthesis in microalgae. Furthermore, the hypothesis that the cell wall-deficient mutant facilitates glycolate excretion was proposed and validated by comparing the glycolate accumulation trends of various Chlamydomonas reinhardtii strains. This study will facilitate the development of microalgae-based biotechnology and shed lights on the continuous advancement of green biomanufacturing for industrial application. [ABSTRACT FROM AUTHOR]

We report the creation of 17 Escherichia coli strains harboring the conjugative plasmid pLCasCureT with a CRISPR-Cas9 system to surgically "cure" the most common plasmids among Enterobacterales species. This approach can create isogenic pairs of strains to study host-plasmid interactions, correlate plasmid genotype and phenotype, and create plasmid-free cloning strains. • To surgically cure plasmids using CRISPR editing. • To create plasmid-less strains with no chromosomal perturbation. • To create isogenic strain pairs to evaluate host-plasmid interactions. • To dissect plasmid encoded antibiotic resistance and other accessory genes. [ABSTRACT FROM AUTHOR]

Tocochromanols, collectively known as Vitamin E, serve as natural lipid-soluble antioxidants that are exclusively obtained through dietary intake in humans. Synthesized by all plants, tocochromanols play an important role in protecting polyunsaturated fatty acids in plant seeds from lipid peroxidation. While the genes involved in tocochromanol biosynthesis have been fully elucidated in Arabidopsis thaliana, Oryza sativa and Zea mays, the genetic basis of tocochromanol accumulation in sweet corn remains poorly understood. This gap is a consequence of limited natural genetic diversity and harvest at immature growth stages. In this study, we conducted comprehensive genome-wide association studies (GWAS) on a sweet corn panel of 295 individuals with a high-density molecular marker set. In total, thirteen quantitative trait loci (QTLs) for individual and derived tocochromanol traits were identified. Our analysis identified novel roles for three genes, ZmCS2 , Zmshki1 and ZmB4FMV1, in the regulation of α-tocopherol accumulation in sweet corn kernels. We genetically validated the role of Zmshki1 through the generation of a knock-out line using CRISPR-Cas9 technology. Further gene-based GWAS revealed the function of the canonical tyrosine metabolic enzymes ZmCS2 and Zmhppd1 in the regulation of total tocochromanol content. This comprehensive assessment of the genetic basis for variation in vitamin E content establishes a solid foundation for enhancing vitamin E content not only in sweet corn, but also in other cereal crops. • Thirteen QTLs associated with tocochromanol traits were identified through GWAS in a sweet corn panel. • Three genes, ZmCS2 , Zmshki1 , and ZmB4FMV1 , were novelly found to regulate α-tocopherol accumulation. • CRISPR-Cas9 knockout of Zmshki1 validated its role in vitamin E biosynthesis. • ZmCS2 and Zmhppd1 , enzymes in tyrosine metabolism, were shown to regulate total tocochromanol content. [ABSTRACT FROM AUTHOR]

[Display omitted] • De novo biosynthesis of 2-PE from glucose in Yarrowia lipolytica is improved. • Co-expression of EcAROGfbr and EcPheAfbr significantly increases 2-PE yield. • Increasing copies of key enzymes and knocking out competitive pathways improve yield. • Enhancing the shikimate pathway removes the precursor metabolic bottleneck. The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROGfbr and EcPheAfbr increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT , ylARO1 , and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/g glu , which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives. [ABSTRACT FROM AUTHOR]

Background: Salmonella enterica serovar Choleraesuis (S. C) is a swine enteric pathogen causing paratyphoid fever, enterocolitis, and septicemia in piglets. S. C is mainly transmitted through the fecal-oral route. Vaccination is an effective strategy for preventing and controlling Salmonella infection. Results: Herein, we used CRISPR-Cas9 technology to knockout the virulence regulatory genes, rpoS, and slyA of S. C and constructed the ∆rpoS , ∆slyA, and ∆rpoS ∆slyA strains. The phenotypic characteristics of the mutant strains remained unchanged compared with the parental wild-type strain. In vivo study, unlike the wild-type strain, the ∆slyA and ∆rpoS ∆slyA strains alleviated splenomegaly, colon atrophy, and lower bacterial loads in the spleen, liver, ileum, and colon. These mutant strains survived in Peyer's patches (PPs) and mesenteric lymph nodes (MLN) for up to 15 days post-infection. Furthermore, the immunization of the ∆rpoS ∆slyA strain induced robust humoral and cellular immune responses. Conclusions: Consequently, vaccination with ∆rpoS ∆slyA conferred a high percentage of protection against lethal invasive Salmonella , specifically S. C , in mice. This study provided novel insights into the development of live-attenuated vaccines against the infection of S. C. • Three mutant Salmonella enterica serovar Choleraesuis (S. C) strains ∆rpoS , ∆slyA , and ∆rpoS ∆slyA were generated. • The mutant S.C. strains remained similar phenotypes with the parental strain but with much lower virulence. • The ∆rpoS ∆slyA S.C strain showed ideal immunity and protective efficacy. [ABSTRACT FROM AUTHOR]

The world population is steadily growing, exerting increasing pressure to feed in the future, which would need additional production of major crops. Challenges associated with changing and unpredicted climate (such as heat waves) are causing global food security threats. Cereal crops are a staple food for a large portion of the world's population. They are mostly affected by these environmentally generated abiotic stresses. Therefore, it is imperative to develop climate-resilient cultivars to support the sustainable production of main cereal crops (Rice, wheat, and maize). Among these stresses, heat stress causes significant losses to major cereals. These issues can be solved by comprehending the molecular mechanisms of heat stress and creating heat-tolerant varieties. Different breeding and biotechnology techniques in the last decade have been employed to develop heat-stress-tolerant varieties. However, these time-consuming techniques often lack the pace required for varietal improvement in climate change scenarios. Genome editing technologies offer precise alteration in the crop genome for developing stress-resistant cultivars. CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/Cas9), one such genome editing platform, recently got scientists' attention due to its easy procedures. It is a powerful tool for functional genomics as well as crop breeding. This review will focus on the molecular mechanism of heat stress and different targets that can be altered using CRISPR/Cas genome editing tools to generate climate-smart cereal crops. Further, heat stress signaling and essential players have been highlighted to provide a comprehensive overview of the topic. • Challenges associated with changing and unpredicted climate (such as heat waves) are causing global food security threats due to cereal crop susceptibility. • Genome editing technologies offer precise alteration in the crop genome for developing heat stress-resistant cultivars. • This review highlights different targets associated with heat stress that can be altered using CRISPR/Cas genome editing tools to generate climate-smart cereal crops. [ABSTRACT FROM AUTHOR]

Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200–1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research. Naturally evolved or engineered photosensory modules have been harnessed for transcriptional control and genetic engineering in space and time by using photons emitting in the wide range of 200–1000 nm. Optogenetic modules can be modularly engineered into host cells to control physiological processes via light-switchable allosteric control, oligomeric transition, protein–protein heterodimerization, and self-cleavage. Optogenetic devices can be integrated with the transcriptional machinery, DNA recombinases, RNA-binding proteins, and CRISPR-based genome-engineering tools to precisely manipulate transcriptional reprogramming, DNA/RNA modifications, genetic recombination, and genome/epigenome editing. Optogenetics can be combined with synthetic biology, biophotonics, and genome-engineering approaches to advance precision medicine and personalized therapies for human diseases. [ABSTRACT FROM AUTHOR]

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD+ was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD+ by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD+ by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial applications. [Display omitted] • Disruption of ldh or pfl redirected carbon flux and reduced the consumption of NADH. • Overexpression of budB or budC amplified carbon flux and increased the generation of NAD+. • Yield of 2,3-BDO was increased in all mutants, and the highest one was increased by 2.55 times. • 2,3-butanediol yields of IAM1183-LPBC increased by 1.88 times in 5-L fermenter. [ABSTRACT FROM AUTHOR]

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation. • Application of CRISPR/Cas9 for improving trait in cattle. • Long-term health monitoring of MSTN-mutated cattle. • Stable germline transmission of MSTN-mutated female or male. • Establishment of effective gene-edited embryo production derived from the semen of a MSTN mutated male via electroporation. [ABSTRACT FROM AUTHOR]

Basidiomycetes can produce various valuable secondary metabolites with bioactivities. However, the low titer of metabolites has greatly limited their application. Currently, shortage of mutant complementation techniques, especially in-situ complementation, impeded the elucidation of their metabolites biosynthesis pathways. In this work, Ganoderma lucidum , a traditional Chinese medicinal basidiomycete, which can produce various bioactive ganoderic acids (triterpenoids), was used; and a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) mediated in-situ complementation system for two functional genes was established. For a disruptant of gl26097 , which encodes a Cys2-His2 (C2H2)-type zinc finger transcription factor involved in calmodulin-calcineurin signaling, one in-situ complemented strain of 8 transformants was obtained,concluded by sequencing confirmation. For the function of gl26097 , both the cell growth and content of four identified individual ganoderic acids (GAs) were restored in the complemented strain compared to the gl26097 disruptant. For a disruptant of cyp5150l8 , which is responsible for transforming lanosterol to a GA, one in-situ complemented strain from 8 transformants was obtained as confirmed by sequencing. The titer of four identified GAs increased significantly in the complemented strain compared to the cyp5150l8 disruptant. To the best of our knowledge, this is the first research report on applying CRISPR-Cas9 to complement mutants in-situ in basidiomycetes. The CRISPR-Cas9 assisted gene complementation technology will be helpful to study gene functions and GA biosynthesis pathway in G. lucidum , which may be also applied to other basidiomycetes. [Display omitted] • CRISPR-assisted gene in-situ complementation was established in basidiomycete. • Two gene disruptants of G. lucidum were complemented via the developed technology. • The work will be helpful to gene function and biosynthetic pathway study in G. lucidum. [ABSTRACT FROM AUTHOR]

Fermentative bacteria, the main microbiota in yogurt, interfere with the detection of unintended bacterial contaminants. The removal of fermentative bacteria and enrichment of unintended bacterial contaminants is a challenging task in bacterial detection. The present study developed a new 16S rRNA-depletion PCR for such enrichment and detection. Specifically, a single-guide RNA was designed and synthesized based on the 16S rRNA sequence of Streptococcus thermophilus , with the highest DNA abundance in the yogurt. The CRISPR-Cas9 system was used to specifically cleave and remove the genomic DNA of the fermentative bacteria, followed by PCR amplification. This method improved the detection sensitivity, simplified the operation steps, and reduced the detection cost of PCR analysis. We also used the 16S rRNA-depletion PCR to amplify and detect the unintended bacterial contaminants in yogurts with shrunken packages and analyzed the underlying reasons to prevent this issue of product quality. [ABSTRACT FROM AUTHOR]

We previously established a method to generate a human iPS cell-derived myeloid cells (iPS-MLs) from a human leukocyte antigen (HLA)-A *24:02 donor, which could differentiate into dendritic cells (DCs) and had a high potential in stimulating T cells. However, for clinical applications, the histoincompatibility between iPS cells and cells in cancer patients has remained as a key challenge. One solution is to construct the iPS cell library covering all HLA haplotypes, but it is excessively laborious and impractical. In order to overcome this problem, we generated HLA-modified iPS cells to avoid immune rejection. We disrupted HLA-A , -B and DRA genes by using a CRISPR-Cas9 system and subsequently introduced HLA-A *02:01 into iPS-MLs. We examined the immunogenic capacities of these HLA-deficient iPS-MLs for inducing antigen-specific CD8+ T cells derived from an allogeneic donor. HLA-deficient iPS-MLs could avoid recognition by alloreactive CD8+ T cells. HLA-deficient iPS-MLs with introduced HLA-A *02:01 differentiated into functional DCs upon stimulation with IL-4, and induced HLA-A2-restricted melanoma antigen recognized by T cells-1 (MART)−1-specific CD8+ T cells derived from an allogeneic donor. Furthermore, HLA-deficient iPS-MLs with introduced HLA-A *02:01 and MART-1 could induce HLA-A2-restricted MART-1-specific CD8+ T cells derived from an allogeneic donor. We developed a method that can exchange HLA alleles from HLA-A *24:02 to* 02:01 in iPS-MLs, and our findings may overcome the obstacle of histoincompatibility in DC vaccination therapy, regardless of HLA allele. [ABSTRACT FROM AUTHOR]

One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the β-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic β-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (β cat Δex3). We then compared β cat Δex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apc fs-ex15 ). We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apc fs-ex15 liver tumors. Both Apc fs-ex15 and βcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1 -mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. Apc fs-ex15 and βcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and β-catenin-activated human HCCs or mesenchymal hepatoblastomas. New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model. [Display omitted] • β-catenin gain-of-function is oncogenic in the liver. • In vivo editing by CRISPR/Cas9 was able to phenocopy Cre-lox models of liver-targeted oncogenesis in mice. • β-catenin gain-of-function is equivalent to Apc loss in the liver. • Both models of β-catenin activation produce differentiated hepatocellular carcinoma. • Both models produce undifferentiated hepatoblastoma-like tumors. [ABSTRACT FROM AUTHOR]

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs. • Blocking the expression of Calpain-3 protein can generate LGMDR1 disease model. • The increase of pulses allows a greater entry of RNP but also greater cell damage. • sgRNA concentration is an important factor for edition in CRISPR electroporation. • Oocyte electroporation is a faster and easier method than microinjection. • Oocyte electroporation achieves similar efficiencies to microinjection. [ABSTRACT FROM AUTHOR]

Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4 -related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD+ modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo , these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction. • Human dermal fibroblasts of African descent were reprogrammed into iPSCs. • CRISPR-Cas9 was used to generate NDUFS4 KO iPSCs. • KO iPSCs display a mitochondrial disease-relevant metabolic phenotype. • Metabolic perturbations are centered around NAD+/NADH redox imbalance. • KO iPSCs provide a model suitable for drug screening in the pluripotent state. [ABSTRACT FROM AUTHOR]

Breeding programs for the tiger puffer (Takifugu rubripes), a prominent species in East Asian aquaculture, are underway to establish aquaculture strains with economically desirable genetic traits. However, their brood stocks have large body sizes and require a long time to reach sexual maturity. Surrogate broodstock technology involves generating donor-derived gametes from surrogate (recipient) fish via germ cell transplantation. This method offers a promising solution to overcome the challenges faced in tiger puffer breeding. This study established a sterilization technique using a CRISPR-Cas9 mediated knockout line in the grass puffer (T. alboplumbeus), which serves as a suitable surrogate due to its smaller body size and shorter generation time compared to the tiger puffer. We evaluated the potential of germ cell–deficient grass puffers as recipients for the efficient production of donor-derived tiger puffer gametes. The mutated alleles were induced in the crispant resulting from the microinjection of genome editing solution containing single guide RNA targeted to dead end 1 (dnd1) into fertilized eggs and inherited by the F1 generation. Homozygous mutants generated in the F2 generation by crossing F1 heterozygous mutants displayed germ cell deficiency. Histological observations and gene expression analysis revealed sexual dimorphism in the germ cell–deficient gonads of dnd1 homozygous mutants. The incorporation of donor cells into the recipient gonads was confirmed via the intraperitoneal transplantation of tiger puffer testicular cells into dnd1 homozygous mutants. Donor-derived tiger puffer gametogenesis was observed histologically, with only donor-derived tiger puffer sperm successfully produced in recipients with dnd1 homozygous mutations at one year of age. Thus, the use of a germ cell–deficient grass puffer prepared by crossing the dnd1 heterozygous mutant line as a recipient to only produce donor-derived gametes improves the efficiency of surrogate production and may enhance the efficiency of the breeding process in tiger puffer aquaculture. • CRISPR Sterilization: Introduced CRISPR-Cas9 to sterilize grass puffers, facilitating tiger puffer surrogate production. • Streamlined Technique: Utilized dnd1 mutants as effective recipients, accelerating xenogeneic gamete production. • Fast-tracking Aquaculture: Leveraged the smaller, faster-generation grass puffer for quicker tiger puffer strain development. • Genetic Resource Management: dnd1 mutants offer a tool for managing genetic resources, ensuring efficiency and preservation. [ABSTRACT FROM AUTHOR]

Cardiac amyloidosis (CA) is a form of infiltrative, restrictive cardiomyopathy that presents a diagnostic and therapeutic challenge in clinical practice. Historically, it has led to poor prognosis due to limited treatment options. However, advancements in disease awareness, diagnostic tools, and management approaches have led to the beginning of an era characterized by earlier diagnosis and a broader range of treatments. This article examines the advances in treating the two primary forms of cardiac amyloidosis: transthyretin cardiac amyloidosis (ATTR-CA) and light chain mediated cardiac amyloidosis (AL-CA). It highlights therapies for ATTR-CA that focus on interrupting the process of amyloid fibril formation. These therapies include transthyretin stabilizers, gene silencers, and monoclonal antibodies, which have shown the potential to improve patient outcomes and survival rates significantly. As of this writing, tafamidis is the sole Food and Drug Administration (FDA)--approved drug for ATTR-CA; however, experts anticipate several other drugs will gain approval within 1–2 years. Treatment strategies for AL-CA typically involve chemotherapy to inhibit the clonal cell type responsible for excessive AL amyloid fibril production. The prognosis for both types of amyloidosis primarily depends on how much the heart is affected, with most deaths occurring due to progressive heart failure. Effective care for CA patients requires collaboration among specialists from multiple disciplines, such as heart failure cardiology, electrophysiology, hematology/oncology, nephrology, neurology, pharmacology, and palliative care. [ABSTRACT FROM AUTHOR]

Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a dependency on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M checkpoint kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here, using CRISPR-Cas9 knockout screens in vitro and in vivo , we identified multiple factors influencing the response of SCLC cells to the WEE1 kinase inhibitor AZD1775, including the GCN2 kinase and other members of its signaling pathway. Rapid activation of GCN2 upon AZD1775 treatment triggers a stress response in SCLC cells. Pharmacological or genetic activation of the GCN2 pathway enhances cancer cell killing by AZD1775. Thus, activation of the GCN2 pathway represents a promising strategy to increase the efficacy of WEE1 inhibitors in SCLC. [Display omitted] • CRISPR-Cas9 screens identify genetic determinants of response to the WEE1 inhibitor AZD1775 • GCN2 inactivation leads to resistance to AZD1775 treatment in vitro and in vivo • GCN2 gets rapidly activated upon WEE1 inhibition by AZD1775 • Activation of the GCN2 pathway sensitizes cells to AZD1775 treatment Drainas et al. identify GCN2 as a factor influencing small-cell lung cancer (SCLC) response to the WEE1 inhibitor AZD1775. AZD1775 directly activates the GCN2 stress response pathway, increasing its effectiveness in killing SCLC cells. Thus, activating the GCN2 pathway presents a novel opportunity to enhance WEE1 inhibitor efficacy in SCLC. [ABSTRACT FROM AUTHOR]

• In this paper, we have developed a novel system termed Assembled PCR & CRISPR for Ultra-sensitive Rifampin-resistant Analysis Testing (ACURAT). • This system is capable of detecting less than 10 copies of TB and specifically identifying fifteen drug-resistant related mutated genotypes. • To overcome the challenge of PAM sequence is missing in almost all mutant codons, ACURAT introduced a PCR-assisted scheme, PAM-PCR, to introduce the PAM sequence into desired positions. • To increase the specificity of CRISPR-Cas9 system, we designed a micro-mismatch CRISPR-Cas9 system, which has a 1 bp mismatch with the wildtype codon, but at least a 2 bp mismatches with the mutant codon. • Finally, we found that 19 nt sgRNA, 1 or 2 bp distance to PAM sequence, and HiFi-Cas9 nuclease could is the best parameters for the ACURAT. Tuberculosis (TB) remains a pressing global health threat, while the emergence of drug-resistant TB adds complexity to clinical treatment strategies, underscoring the need for rapid and accurate diagnosis. In this study, we present an innovative identification and antibiotic susceptibility testing system, termed Assembled PCR & CRISPR for Ultra-sensitive Rifampin-resistant Analysis Testing (ACURAT). ACURAT employed a nested polymerase chain reaction (PCR) scheme to sensitively detect as few as 10 copies of the TB genome, more importantly, addressing the clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR-Cas9) system's requirement for a protospacer adjacent motif (PAM) sequence by introducing with the designed primers. Recognizing the limitation of fully-matched guide ribonucleic acid (RNA) in distinguishing one-base single nucleotide polymorphisms (SNPs), we designed a micro-mismatch CRISPR-Cas9 system with significant selectivity, featuring a single-base mismatch with wildtype genotypes and at least two base mismatches with drug-resistant genotypes. The systematic optimization of ACURAT parameters revealed that a 19 nt guide RNA length, a 1 bp distance to the PAM sequence, and the use of a high-fidelity Cas9 nuclease significantly enhanced the efficiency. Finally, ACURAT successfully distinguished fifteen drug-resistance-related genotypes in codons 516, 526, 531, and 533 of the polymerase beta subunit gene. ACURAT demonstrated substantial clinical potential for efficient TB diagnosis and exhibited the capability to address other sequence recognition scenarios. [ABSTRACT FROM AUTHOR]

Silk is a natural protein fiber that is predominantly comprised of fibroin and sericin. In addition, it contains seroins, protease inhibitors, enzymes, and other proteins. We found an ecdysone oxidase BmGMC2, notably, which is specifically and highly expressed only in the silk glands of silkworms (Bombyx mori L.). It is also one of the main components of non-cocoon silk, however, its precise function remains unclear. In this study, we examined the spatiotemporal expression pattern of this protein and obtained a homozygous mutant strain (K-GMC2) using the CRISPR-Cas9 system. Compared to the wild-type strain (WT), the silk production and main silk proteins significantly decreased in the larval stage, and the adhesive strength of native silk proteins decreased in the final instar. Proteomic data indicated the abundance of ribosomal proteins decreased significantly in K-GMC2 , differentially expressed proteins (DEPs) were enriched in pathways related to neurodegenerative diseases and genetic information processing, indicating that knockout may lead to a certain degree of cell stress, affecting the synthesis of silk proteins. This study investigated the expression pattern and gene function of ecdysone oxidase BmGMC2 in silk and silk glands, laying the groundwork for understanding the role of enzymes in the production of silk fibers. [Display omitted] • BmGMC2 is the most abundant enzyme in the non-cocoon silk. • The BmGMC2 homozygous mutant strain was obtained using CRISPR-Cas9 system. • Lack of BmGMC2 leads to cellular stress and affects the synthesis of silk proteins. • BmGMC2 affects the adhesion strength of silk proteins and silk production. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9's remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies. • DNA double-strand breaks are severe threats to genomic integrity. • Several mechanisms exist to repair DNA double-strand breaks. • The choice of repair mechanism for DNA double-strand breaks depends on DNA end resection. • A site-specific endonuclease, Cas9 aids in understanding DNA double-strand break repair mechanisms. • Comprehensive understanding of DNA DSB repair supports therapeutic strategies for gene therapy and cancer treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles − extracellular vesicles and virus-like particles − for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene editing cargos and their potential for clinical translation. [ABSTRACT FROM AUTHOR]

• Endogenous circRNAs have been found to have diverse physiological functions, including miRNA/protein sponges and cell activity modulators. • The use of antisense RNA approaches has facilitated the understanding of the different roles of circRNAs. • CircRNAs hold promise as potential targets for therapeutic intervention. Circular RNAs (circRNAs) are a type of noncoding RNA that are formed by back-splicing from eukaryotic protein-coding genes. The most frequently reported and well-characterized function of circRNAs is their ability to act as molecular decoys, most often as miRNA and protein sponges. However, the functions of most circRNAs still need to be better understood. To more fully understand the biological relevance of validated circRNAs, knockdown functional analyses can be performed using antisense oligonucleotides, RNA interference (RNAi) experiments (e.g., targeting back-splicing junction sites), the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)-9 system (e.g., generating circRNA-specific knockouts), and CRISPR-Cas13 technology to effectively target circRNAs without affecting host genes. In this review, I summarize the feasibility and effectiveness of circRNA knockdown through antisense strategies for investigating the biological roles of circRNAs in cultured cells and animal models. [ABSTRACT FROM AUTHOR]

Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals. [Display omitted] • G3BP1-GFP stress granule reporter introduced in SK-N-SH cells via CRISPR/Cas9. • Stress granule response to BPA validated in G3BP1-GFP reporter cells. • Established cell line mimics stress granule response of neuroblastoma. • Reporter system offers a tool to study neuronal stress and stress granule dynamics. [ABSTRACT FROM AUTHOR]

• NCLIV_038210 -encoded protein is a novel dense granule protein, named NcGRA85. • Deletion of the NcGRA61 and NcGRA85 does not affect N. caninum growth and virulence. • NcGRA27 is important for the growth and virulence of N. caninum. • NcGRA27 is most likely a new virulence factor of N. caninum. Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum. [ABSTRACT FROM AUTHOR]

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism. [Display omitted] • Single double-strand break causes transcription repression of broken genes in a few minutes • Transcription repression propagates along the chromosome for hundreds of kilobases • Rapid transcription repression is not regulated by PRC1-mediated H2A K119 ubiquitination • Proteasome-mediated RNAPII removal contributes to transcription repression and propagation He et al. apply light-activated CRISPR to induce a DNA double-strand break and observe rapid transcription repression and its propagation along the damaged chromosome. They demonstrate that proteasomes remove RNAPII around the damaged site. Their method builds a foundation to measure the kinetics after DSB and elucidate the molecular mechanism. [ABSTRACT FROM AUTHOR]

Single nucleotide variants (SNVs) constitute the most frequent variants that cause human genetic diseases. Base editors (BEs) comprise a new generation of CRISPR-based technologies, which are considered to have a promising future for curing genetic diseases caused by SNVs as they enable the direct and irreversible correction of base mutations. Two of the early types of BEs, cytosine base editor (CBE) and adenine base editor (ABE), mediate C-to-T, T-to-C, A-to-G, and G-to-A base transition mutations. Together, these represent half of all the known disease-associated SNVs. However, the remaining transversion (i.e., purine–pyrimidine) mutations cannot be restored by direct deamination and so these require the replacement of the entire base. Recently, a variety of base transversion editors were developed and so these add to the currently available BEs enabling the correction of all types of point mutation. However, compared to the base transition editors (including CBEs and ABEs), base transversion editors are still in the early development stage. In this review, we describe the basics and advances of the various base transversion editors, highlight their limitations, and discuss their potential for treating human diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

The energy sector faces significant challenges globally, including increasing air pollution, rising energy demands, and fluctuating crude oil prices. In response, researchers have turned their attention to third-generation biofuels derived from algae due to their numerous advantages over first (crop-based) and second-generation (petro crop-based) biofuels. Microalgae offer faster growth rates, shorter regeneration times, the potential to utilize waste lands for cultivation, and scalability. However, challenges such as maintaining biomass yield while increasing lipid synthesis persist. To address this challenge, researchers are employing metabolomic strategies to target the lipid production pathway in microalgae. This review provides a comprehensive overview of various metabolomic approaches, including gene editing techniques such as CRISPR-Cas9, RNA interference (RNAi), zinc finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENs), aimed at enhancing lipid synthesis while maintaining biomass yield in microalgae. Additionally, the review discusses major challenges and the future potential of microalgal-based biofuels. • Comprehensive reviews of several research on the generation of biofuel from microalgae algae are included. • oThe best methods for producing biofuel from microalgae are reviewed. • oFuture potential and current issues with producing biofuel from microalgae algae are discussed. [ABSTRACT FROM AUTHOR]

Objective: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes.Method: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140-/- mice.Results: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium.Conclusion: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance. [ABSTRACT FROM AUTHOR]

Therapy resistance associated with relapse is a main cause of death in acute myeloid leukemia (AML). To address this issue, a dual-targeting CRISPR-Cas9 genome editing nanosystem was constructed for CXCR4 knockout to reverse the malignancy of leukemia cells. The surface of the dual-targeting nanosystem is composed of MUC1 specific aptamer incorporated alginate (MUC1 aptamer-alginate) and T22-NLS peptide with T22 sequence targeting CXCR4; the core of the nanosystem consists of protamine complexed with CRISPR-Cas9 plasmid. The in vitro study shows that the nanosystem mediated genome editing induces cell apoptosis, cell cycle arrest, as well as inhibited cell migration and adhesion in edited THP-1 cells after CXCR4 knockout. Further, the unprocessed peripheral blood from acute myeloid leukemia (AML) patients was directly used to carry out ex vivo study. The results show the genome editing nanosystem can effectively knock out CXCR4 in leukemia cells, leading to attenuated CXCR4 protein as studied by antibody labeling and reduced CXCR4 mRNA as probed by a molecular beacon delivery system. In addition to developing a promising delivery vector for gene therapy on AML, this study also provides an effective strategy to evaluate the therapeutic efficiency of particular treatments by peripheral blood-based ex vivo studies. [Display omitted] [ABSTRACT FROM AUTHOR]

• Colorectal carcinoma associated with proficient MLH1 gene are more aggressive cancers. • MLH1 influences the chemoresistance by mediating nuclear autophagy, nucleophagy. • Interaction among MLH1 and autophagy marker LC3 facilitated nucleophagy induction. • MLH1 mediated nucleophagy determined chemoresistance in colorectal cancer cells. [Display omitted] Colorectal Cancer (CRC) with Microsatellite instability (MSI) and mutLhomolog-1 (MLH1) gene deficiency are less aggressive than MLH1 proficient cancers. MLH1 is involved in several cellular processes, but its connection with the autophagy-dependent cellular response towards anticancer drugs remains unclear. In this study, we aimed to investigate the interaction between MLH1 and the autophagy marker LC3, which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-Fluorouracil (5-FU) induced cell death. To examine the role of MLH1 in DNA-damage-induced nucleophagy in CRC cells, we utilized a panel of MLH1 deficient and MLH1 proficient CRC cell lines. We included a parental HCT116 cell line (MLH1−/−) and its isogenic cell line HCT116 MLH1 +/− in which a single allele of the MLH1 gene was introduced using CRISPR-Cas9 gene editing. We observed that MLH1 proficient cells were less sensitive to the 5-FU-induced cytotoxic effect. The 5-FU induced DNA damage led to LC3 up-regulation, which was dependent on MLH1 overexpression. Moreover, immunofluorescence and immunoprecipitation data showed LC3 and MLH1 were colocalized in CRC cells. Consequently, MLH1 dependent 5-FU-induced DNA damage contributed to the formation of micronuclei. These micronuclei colocalize with autolysosome, indicating a cytoprotective role of MLH1 dependent nucleophagy. Interestingly, siRNA knockdown of MLH1 in HCT116 MLH1 +/− prevented LC3 upregulation and micronuclei formation. These novel data are the first to show an essential role of MLH1 in mediating the chemoresistance and survival of cancer cells by increasing the LC3 expression and inducing nucleophagy in 5-FU treated CRC cells. [ABSTRACT FROM AUTHOR]

The discovery, only a decade ago, of the genome editing power of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases is already reinventing the therapeutic process, from how new drugs are discovered to novel ways to treat diseases. CRISPR-based screens can aid therapeutic development by quickly identifying a drug's mechanism of action and escape mutants. Additionally, CRISPR-Cas has advanced emerging ex vivo therapeutics, such as cell replacement therapies. However, Cas9 is limited as an in vivo therapeutic due to ineffective delivery, unwanted immune responses, off-target effects, unpredictable repair outcomes, and cellular stress. To address these limitations, controls that inhibit or degrade Cas9, biomolecule–Cas9 conjugates, and base editors have been developed. Herein, we discuss CRISPR-Cas systems that advance both conventional and emerging therapeutics. CRISPR screens can advance conventional drug development by identifying key targets and escape mutants. CRISPR-based therapies are rapidly advancing, with applications involving cell replacement therapies, gene drives, and diagnostics. The use of CRISPR both ex vivo and in vivo is hindered by the immunogenicity of CRISPR components, limited delivery, low specificity, off-target effects, DNA damage, and repair pathways. CRISPR limitations are being addressed through advances in controllers (degraders/inhibitors) of Cas9, conjugation strategies to endow Cas9 with new functions, and base and primer editors that do not produce double-strand breaks. [ABSTRACT FROM AUTHOR]