CRISPR-Cas9 System Research Articles

Pseudopregnant mice generated from Piwil1 deficiency sterile mice.

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.

Functional characterization of three novel dense granule proteins in Neospora caninum using the CRISPR-Cas9 system

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.

P09 Next-generation sequencing: on-and off-target analysis of gene editing in fibroblasts from patients with recessive dystrophic epidermolysis bullosa

Abstract Introduction and aims Base editors (BEs) and prime editors (PEs) have proved to have advantages over traditional clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in correcting pathogenic mutations. We previously demonstrated 94.6% efficiency using adenine base editor (ABE)8e in correcting nonsense mutation (c.5047 C>T) in fibroblasts, which causes recessive dystrophic epidermolysis bullosa (RDEB). However, safety profile and cell-type-specific efficiencies of gene editing techniques have not been characterized. The aim of our research is to establish corrections of c.5047 C>T pathogenic variant in fibroblasts and compare off-target effects and efficiency of CRISPR-Cas9, BE and PE systems. Methods Primary fibroblasts harbouring heterozygous C>T pathogenic variant on exon 54 will be transfected with CRISPR-Cas9, ABE8e and PE using electroporation or lipofectamine. Both Sanger sequencing and next-generation sequencing (NGS) will be conducted to confirm pathogenic variant corrections. Galaxy platform which incorporates different NGS analysis tools will be used to analyse the NGS data. Subsequently, whole-exome sequencing and targeted amplicon sequencing will be used for off-target analysis. Results On-target efficiency for ABE8e to correct c.5047 C>T pathogenic variant in patient primary fibroblasts was determined first by Sanger sequencing showing 100% editing in contrast to 84% determined by NGS. Moving forward, we plan to correct the same pathogenic variant with PEs. Initially experiments are under way with PEmax mRNA and engineered PE guide RNAs (epegRNA) to compare different PEs. Furthermore, we will investigate the off-target effects of CRISPR-Cas9, ABE8e and PEs to edit RDEB primary fibroblasts using NGS Methods and conduct functional studies. Conclusions Here, we showed that the c.5047 C>T pathogenic variant in RDEB fibroblasts could be successfully edited using ABE8e. We demonstrated that NGS is required to precisely determine the editing efficacy. Taken together, we have established a pipeline for base editing in RDEB and we aim to achieve the same for prime editing in the future.

Increasing the efficiency of gene editing with CRISPR-Cas9 via concurrent expression of the Beta protein

Escherichia coli has emerged as an important host for the production of biopharmaceuticals or other industrially relevant molecules. An efficient gene editing tool is indispensable for ensuring high production levels and optimal release of target products. However, in Escherichia coli, the CRISPR-Cas9 system has been shown to achieve gene modifications with relatively low frequency. Large-scale PCR screening is required, hindering the identification of positive clones. The beta protein, which weakly binds to single-stranded DNA but tightly associates with complementary strand annealing products, offers a promising solution to this issue. In the present study, we describe a targeted and continuous gene editing strategy for the Escherichia coli genome. This strategy involves the coexpression of the beta protein alongside the CRISPR-Cas9 system, enabling a variety of genome modifications such as gene deletion and insertion with an efficiency exceeding 80 %. The integrity of beta proteins is essential for the CRISPR-Cas9/Beta-based gene editing system. In this work, the deletion of either the N- or C-terminal domain significantly impaired system efficiency. Overall, our findings established the CRISPR-Cas9/Beta system as a suitable gene editing tool for various applications in Escherichia coli.

CRISPR-Cas9: An Evolutionary Technique for the Treatment of Breast and Cervical Carcinoma

CRISPR/Cas9 has transformed genome editing methods in several scientific domains, including the study of human cancer. Globally, breast cancer (BC) affects women more than any other kind of cancer and triple-negative breast cancer (TNBC) has the greatest fatality rate. The diagnosis and treatment of breast cancer have come a long way, yet many patients still experience metastases or relapses. One of the potential techniques for correcting faulty genes and curing different cancers is gene therapy. Certain mutations associated with cancer onset and development might be repaired in model organisms as a first step toward translational applications using the CRISPR/Cas9 technology. The CRISPR/Cas9 system, which consists of a single readily modified guide RNA (sgRNA) sequence coupled to a Cas9 nuclease, has transformed genome editing owing to its simplicity and effectiveness when compared to previous methods. CRISPR/Cas9 allows for the knockdown of over-expressed genes, the reversal of mutations in defected genes, and the remodeling of the regulatory environment to inhibit BC proliferation. CRISPR/Cas9-based treatment mixed with traditional chemotherapy has proven to be beneficial in combating drug resistance and tumor inhibition difficulties. The CRISPR cas9 system may edit many genes at the same time by generating numerous sgRNAs that target different genomic regions, considerably increasing its therapeutic potential and making it a unique technique for treating cervical lesions. This review focuses on the therapeutic applications of CRISPR/Cas9 in the treatment of cervical cancer and breast cancer.

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Designing molecules: directing stem cell differentiation.

Stem cells have been widely applied in regenerative and therapeutic medicine for their unique regenerative properties. Although much research has shown their potential, it remains tricky in directing stem cell differentiation. The advancement of genetic and therapeutic technologies, however, has facilitated this issue through development of design molecules. These molecules are designed to overcome the drawbacks previously faced, such as unexpected differentiation outcomes and insufficient migration of endogenous or exogenous MSCs. Here, we introduced aptamer, bacteriophage, and biological vectors as design molecules and described their characteristics. The methods of designing/developing discussed include various Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedures, in silico approaches, and non-SELEX methods for aptamers, and genetic engineering methods such as homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), Bacteriophage Recombineering with Infectious Particles (BRIP), and genome rebooting for bacteriophage. For biological vectors, methods such as alternate splicing, multiple promoters, internal ribosomal entry site, CRISPR-Cas9 system and Cre recombinase mediated recombination were used to design viral vectors, while non-viral vectors like exosomes are generated through parental cell-based direct engineering. Besides that, we also discussed the pros and cons, and applications of each design molecule in directing stem cell differentiation to illustrate their great potential in stem cells research. Finally, we highlighted some safety and efficacy concerns to be considered for future studies.

Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system

Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Harnessing noncanonical crRNA for highly efficient genome editing

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.

CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Modulation of Cardiac Ryanodine Receptors by Chloride Intracellular Ion Channel 2

Pathogenic variants of the Chloride intracellular ion channel 2 (CLIC2) are implicated in hypertrophic cardiomyopathy (HCM), a diverse heart condition leading to arrhythmic sudden cardiac death and heart failure. CLIC2 is a unique class of metamorphic protein, related to the enzymatic glutathione s-transferase family and form functional ion channel. Artificial bilayer studies have shown that CLIC2 negatively modulates the activity of cardiac ryanodine receptor 2 (RyR2), implicating the sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ release. However, the subsequent consequences of RyR2 and CLIC2 interaction in cardiac physiology remains unknown. Therefore, we ablated clic2 in human induced pluripotent stem cell (hiPSC) line using the CRISPR-Cas9 system and performed cardiomyocyte differentiation. clic2−/− hiPSC-derived cardiomyocytes (hiPSC-CMs) showed altered electrophysiological properties leading to impaired excitation-contraction coupling. clic2−/− hiPSC-CMs showed an increase in beat rate and field potential duration by 143.49±5.96% and 162.43±5.82%, respectively, whereas conduction velocity and spike amplitude decreased by 43.81±2.22% and 43.32±3.54%, respectively compared to WT hiPSC-CMs. Additionally, action potential duration measurements of clic2−/− hiPSC-CMs showed a decrease in APD30, APD50, and APD90 by 74.42%, 65.19%, and 66.46%, respectively compared to WT hiPSC-CMs. Moreover, clic2−/− hiPSC-CMs showed altered Ca2+ handling, with a decrease in peak amplitude (57.07±2.25%) and duration (38.29±1.30%) compared to the WT hiPSC-CMs. The caffeine induced Ca2+ release in clic2−/− hiPSC-CMs, attributed to the SR/ER Ca2+ overload which decreased by 72.19±3.51% compared to WT hiPSC-CMs. Transcriptomics analysis of clic2−/− hiPSC-CMs revealed 223 transcripts with significant changes in expression levels (89 upregulated and 134 downregulated), implicating CLIC2 in hypertrophy cardiomyopathy, dilated cardiomyopathy, and other heart diseases. Furthermore, the protein expression of CLIC2 increases in end-stage failing human heart samples compared to the non-failing heart. In conclusion, our study sheds light on the novel role of CLIC2 in the regulation of cardiac (patho)physiology. National Institutes of Health (NIH) (HL133050, and HL157453); American Heart Association-Transformational Project Award; National Center for advancing translational sciences (TR004178, understudied proteins and druggable targets) (HS). Presidential Predoctoral Fellowship; Margaret T. Nishikawara Merit Scholarship Endowment in Physiology; Graduate School’s Alumni Grants for Graduate Research and Scholarship Program, The Ohio State University (SS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ZmASR1 negatively regulates drought stress tolerance in maize

Abscisic acid-, stress-, and ripening-induced (ASR) proteins in plants play a significant role in plant response to diverse abiotic stresses. However, the functions of ASR genes in maize remain unclear. In the present study, we identified a novel drought-induced ASR gene in maize (ZmASR1) and functionally characterized its role in mediating drought tolerance. The transcription of ZmASR1 was upregulated under drought stress and abscisic acid (ABA) treatment, and the ZmASR1 protein was observed to exhibit nuclear and cytoplasmic localization. Moreover, ZmASR1 knockout lines generated with the CRISPR–Cas9 system showed lower ROS accumulation, higher ABA content, and a higher degree of stomatal closure than wild-type plants, leading to higher drought tolerance. Transcriptome sequencing data indicated that the significantly differentially expressed genes in the drought treatment group were mainly enriched in ABA signal transduction, antioxidant defense, and photosynthetic pathway. Taken together, the findings suggest that ZmASR1 negatively regulates drought tolerance and represents a candidate gene for genetic manipulation of drought resistance in maize.

Cytokinin-deficient Chlamydomonas reinhardtii CRISPR-Cas9 mutants show reduced ability to prime resistance of tobacco against bacterial infection.

Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.

CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.

CRISPR-based editing strategies to rectify EYA1 complex genomic rearrangement linked to haploinsufficiency

Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.

The Construction of CRISPR-Cas9 System Targeting Vector for CYP3A4 Gene in Hepatic Cell Lines

Drug metabolism in the human liver initiated by Cytochrome P450 (CYP450) has been widely acknowledged. They oxidise drugs, harmful compounds, and endogenous molecules like steroids. In our research study, we mainly focused on CYP3A4 of the CYP450 subfamily that is widely available in the liver around 15% - 20% of hepatic content and plays a significant function in metabolising up to 50% of drugs in the market today. However, the CYP3A4 gene encodes CYP3A4 enzymes that are highly polymorphic, which could affect enzymatic activity and cause variable responses in drug metabolism. Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 also known as the CRISPR-Cas9 system is a gene editing method that allows scientists to edit parts of the genome by insertions and deletions. Hence, this method would be useful for genetic variants such as the CYP3A4 gene. The objectives of this study are to design a guide RNA (gRNA), to insert the designed gRNA into pGuide-it-Zs-Green1 vector, to quantify the purified CYP3A4-KO plasmid vector by using NanoDrop® and to transfect the constructed vector in a hepatic cell line. The methodology involved the selection of the gRNA by using the online gene editing tool, Synthego (https://design.synthego.com), annealing oligos of the gRNA for CYP3A4, cloning gRNA into a plasmid vector, isolation, and purification of the CYP3A4-KO plasmid vector. The construction of the CRISPR-Cas9 targeting vector in this study was successfully achieved and promising since the selected gRNA for CYP3A4 gene which is 5’-ATAAATCCCACTGGACCAAA-3’ and located in exon 5 was correctly ligated after the confirmed with sequencing reaction and cloned it into a plasmid vector. The yield of pCYP3A4-KO plasmid DNA was a good candidate for transfection.

Combined Transcriptome and Proteome Analysis Reveals the Molecular Mechanism by Which ZmPDI Improves Salt Resistance in Rice (Oryza sativa)

As one of the most salt-tolerant grasses, characterizing salt-tolerance genes of Zoysia matrella [L.] Merr. not only broaden the theoretical information of salt tolerance, but also provide new salt-resistant genetic resources for crop breeding. The salt-inducible protein disulfide isomerase (ZmPDI) of Zoysia matrella [L.] Merr. was proved to enhance salt tolerance in homologous overexpression transgenic plants. In order to evaluate its potential application in crops, we conducted the salt tolerance evaluation in heterologous overexpression transgenic rice (OX-ZmPDI), Wild-type (WT) rice, and LOC_Os11g09280 (OsPDI, homologous gene of ZmPDI in rice) knock-out rice generated by CRISPR-Cas9 system (CR-OsPDI). Our findings revealed that OX-ZmPDI rice was higher and exhibited longer main root length, more proline (Pro) and malondialdehyde (MDA) and peroxidase (POD) activity than WT control after salt treatment, while CR-OsPDI resulted in contrary phenotypes. These results indicated that ZmPDI can significantly enhance the salt tolerance in rice, whereas loss-of-function of OsPDI reduces the salt tolerance. To further investigate these differences at the molecular level, we collected roots from OX-ZmPDI transgenic, CR-OsPDI transgenic, and wild-type (WT) plants at 0 and 24 h after salt treatment for RNA-seq and data-independent acquisition (DIA) proteome sequencing. Combined analysis of the transcriptome and proteome revealed that ZmPDI has the potential to enhance the salt tolerance of rice by modulating the expression of laccase-6, zingipain-2, WIP3, FKBP65, AKR4C10, GBSSII, Pho1, and TRXf1. Those results provided new information for the molecular regulation mechanism by which ZmPDI improves salt tolerance, and prove the potential of ZmPDI for application in crop breeding.

Abstract P23: Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction

Abstract Mitochondria is the main power supply for cellular activity and a key player for the regulation of cellular metabolism, and they contain their own DNA. Critical proteins for mitochondrial functions are not only encoded by nuclear DNA (nDNA) but also by mitochondrial DNA (mtDNA). Abnormalities in mitochondrial functions could lead to diseases including neurodegeneration, diabetes, and cancer progression. The ability for a non-coding RNA (β2.7) of human cytomegalovirus (CMV) to localize to the host mitochondria revealed its potential to be used as a tool for mitochondria gene targeting. Using computational methods, our lab identified four thermodynamically conserved structural subdomains (D1 to D4) within the β2.7 RNA. While all four domains exhibited mitochondrial uptake potential, sub-domain D3 exhibited the highest absolute mitochondrial targeting activity and sub-domain D2 the highest activity per nucleotide. Further modifications on different sub-domain alignments found that maximum uptake efficiency was achieved by tandem repeats of 4 copies of D3 domains followed by 4 copies of D2 domains (abbreviated as D3 × 4_D2 × 4). Delivery of a GFP-coding mRNA or antisense RNA targeting mitochondrial genes mt-ATP6 and mt-ATP8 both confirmed its ability to co-deliver functional RNA into mitochondria. To cleave and destroy mutated mitochondrial genomes for future gene therapeutic applications, we explore mitochondrial delivery of the CRISPR-Cas9 system. The mRNAs coding for the wild-type Cas9 as well as more specific Cas9 variants were fused to the mitochondrial delivery vector (D3 × 4_D2 × 4). Latest RT-qPCR on mitochondrial RNA showed delivery of Cas9 mRNA into mitochondria of HepG2 cells. Currently, we investigate co-delivery of single-guide RNA and quantify Cas9 expression in mitochondria. In an alternative approach we deliver sequences coding for functional mitochondrial protein. Examples are the mRNAs coding for mitochondrial proteins mt-ND1, -ND4 or -ND6 which are frequently mutated is patients with Leber’s hereditary optic neuropathy (LHON). Citation Format: Jinglin Ruan, Volker Patzel. Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P23.

CAMSAP3-mediated regulation of HMGB1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation

BackgroundDeregulation of cell death is a common characteristic of cancer, and resistance to this process often occurs in lung cancer. Understanding the molecular mechanisms underlying an aberrant cell death is important. Recent studies have emphasized the involvement of calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) in lung cancer aggressiveness, its influence on cell death regulation remains largely unexplored. MethodsCAMSAP3 was knockout in lung cancer cells using CRISPR-Cas9 system. Cell death and autophagy were evaluated using MTT and autophagic detection assays. Protein interactions were performed by proteomic analysis and immunoprecipitation. Protein expressions and their cytoplasmic localization were analyzed through immunoblotting and immunofluorescence techniques. ResultsThis study reveals a significant correlation between low CAMSAP3 expression and poor overall survival rates in lung cancer patients. Proteomic analysis identified high mobility group box 1 (HMGB1) as a candidate interacting protein involved in the regulation of cell death. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) resulted in increased HMGB1 acetylation and its translocation to the cytoplasm and secretion, thereby inducing autophagic cell death. However, this process was diminished in CAMSAP3 knockout lung cancer cells. Mechanistically, immunoprecipitation indicated an interaction between CAMSAP3 and HMGB1, particularly with its acetylated form, in which this complex was elevated in the presence of TSA. ConclusionsCAMSAP3 is prerequisite for TSA-mediated autophagic cell death by interacting with cytoplasmic acetylated HMGB1 and enhancing its release. SignificantThis finding provides molecular insights into the role of CAMSAP3 in regulating cell death, highlighting its potential as a therapeutic target for lung cancer treatment.