Recent advances in CRISPR-based genome editing technology and its applications in cardiovascular research

The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.

Germline Gene-Editing Creates Enhanced Livestock-Technical and Especially Ethical Issues Challenge Its Use in Humans

Using clustered regularly interspaced short palindromic repeats(CRISPR)-based molecular tools,scientists are engineering-as they are also doing with plants.-animals with advantageous traits,like disease resistance and improved food yield.While these innovative techniques could one day be applied in humans,technical hurdles and ethical concerns likely place this possibility far in the future,The enhancements rely on germline gene editing,which alters the genes in a way that passes the changes on to offspring.Ger m-line gene editing differs from the somatic cell gene editing used in the highly promising new treatment recently approved for the human disease sickle cell anemia.

A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

New insights into ATR inhibition in muscle invasive bladder cancer:The role of apolipoprotein B mRNA editing catalytic subunit 3B

Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in‘Feizixiao'litchi

Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

A bibliometric analysis of gene editing research

Gene editing, as a breakthrough in life science and medicine, makes significant scientific impact. The entire development process of gene editing through bibliometric analysis is studied to explore the distribution features and research focus with visual tool. The number of annual publications distribution, journal distribution and country distribution are analyzed in the basic distribution analysis. The keywords and co-citation are analyzed with CiteSpace to explore the research hotspots. The number of publications related to gene editing is increasing year by year. USA is the main publishing countries for gene editing research. CRISPR/Cas system is a hot topic in the field of genetic editing in last few years. Gene editing is opening up a world of possibilities for the treatment of genetic diseases.

Cre-recombinase systems for induction of neuronspecific knockout models:a guide for biomedical researchers

Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respectively.Using these technologies in mouse embryos led to the generation of mouse knocko ut models and many scientific discoveries.The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as cluste red regula rly inters paced short palindromic repeats for targetted gene deletion.However,several limitations including unwanted off-target gene deletion have hindered their widespread use in the field.Crerecombinase technology has provided additional capacity for cell-specific gene deletion.In this review,we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes.This article has been constructed to provide some background info rmation for the new trainees on the mechanism and to provide necessary information for the design,and application of the Cre-recombinase system thro ugh reviewing the most f requent promoters that are currently available for genetic manipulation of neuro ns.We additionally will provide a summary of the latest technological developments that can be used for targeting neurons.This may also serve as a general guide for the selection of appropriate models for biomedical research.

Development and Application of Prime Editing in Plants

Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)-mediated genome editing has greatly accelerated progress in plant genetic research and agricultural breeding by enabling targeted genomic modifications.Moreover,the prime editing system,derived from the CRISPR/Cas system,has opened the door for even more precise genome editing.Prime editing has the capability to facilitate all 12 types of base-to-base conversions,as well as desired insertions or deletions of fragments,without inducing double-strand breaks and requiring donor DNA templet.In a short time,prime editing has been rapidly verified as functional in various plants,and can be used in plant genome functional analysis as well as precision breeding of crops.In this review,we summarize the emergence and development of prime editing,highlight recent advances in improving its efficiency in plants,introduce the current applications of prime editing in plants,and look forward to future prospects for utilizing prime editing in genetic improvement and precision molecular breeding.

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding,offering enormous potential for crop improvement and food production.Although the direct delivery of Cas9-single guide RNA(sgRNA)ribonucleoprotein(RNP)complexes to grapevine(Vitis vinifera)protoplasts has been shown before,the regeneration of edited protoplasts into whole plants has not been reported.Here,we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus.As proof of concept,a single-copy green f luorescent protein reporter gene(GFP)in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts.CRISPR/Cas9 activity,guided by two independent sgRNAs,was confirmed by the loss of GFP f luorescence.The regeneration of GFP−protoplasts into whole plants was monitored throughout development,confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls.We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts,helping to address the regulatory concerns related to genetically modified plants.This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

c-Myc Knockout as a Model for Gene Editing for Training Healthcare Professional Students

Correction of genetic errors, commonly known as gene editing, holds promise to treat diseases with unmet medical needs. However, gene therapy trials do encounter unwanted outcomes, because of an incomplete understanding of the disease states, and gene therapy processes, among others. This situation encourages a concept that healthcare professionals receiving laboratory research training will not only identify inadequacies in basic biomedical knowledge of gene therapies but also provide tangible refinements. To this end, we have undertaken the PharmD student training in gene editing in a basic research laboratory setting. As a model, MYC gene was chosen for knockout using CRISPR-Cas9 method in HT29 and OVCAR8 cells. Students were involved in the design of MYC-specific gRNAs, subcloning into Cas9-carrying plasmid, and selection of knockout clones from the transfected cells. Subsequently, genomic DNA isolation and sequencing, analysis of clonal DNA sequences using online bioinformatics tools, western blotting, cell proliferation and cell division cycle experiments, were performed to characterize the MYC knockout clones. Results presented in this communication suggest that healthcare professionals who received laboratory training gain a better understanding of the disease states and mechanisms, gene therapy protocols, limitations of gene therapies, ability to critically evaluate the literature and confidence in the oversight of gene therapies in the clinic.

Mitochondrial RNA editing sites affect the phylogenetic reconstruction of gymnosperms

RNA editing is a post-transcriptional process that alters the genetic information of mRNAs,so that the translated proteins deviate from those predicted by the genomic DNA templates.RNA editing occurs in a wide range of eukaryotes,including protists,multicellular animals,and land plants(Gray,2012;Ichinose and Sugita,2016).RNA editing in animals,dominated by A-to-I synonymous editing with low editing efficiency(20e30%),might play important roles in the regulation of gene expression(Wang et al.,2013).In contrast,RNA editing in plant organelles is usually nonsynonymous,affecting mostly the 2nd and 1st codon positions with very high editing efficiency(~80%).In plants,RNA editing is thought to affect plant phenotype and growth by restoring evolutionarily conserved amino acids and ensuring the correct folding of trans-membrane proteins on the respiratory chain complex(Sloan,2017).

Os DXR interacts with Os MORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors. In this study, we identified OsDXR, a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR is a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type.Moreover, we found that the RNA editing efficiencies of ndhA-1019 and rpl2-1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. Finally, disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway resulted in defects in chloroplast development and the RNA editing of chloroplast genes.

See the color,see the seed:GmW1 as a visual reporter for transgene and genome editing in soybean

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.

Multiplex gene editing reduces oxalate production in primary hyperoxaluria type 1

Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening type of primary hyperoxaluria.The compact Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)from the Prevotella and Francisella 1(Cpf1)protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus(AAV)delivery.We hypothesized that the multiplex capabilities of the Cpf1system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1(Hao1)and lactate dehydrogenase A(Ldha)genes.Study cohorts included treated PH1 rats(Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age),phosphate-buffered saline(PBS)-injected PH1 rats,untreated PH1 rats,and age-matched wild-type(WT)rats.The most efficient and specific CRISPR RNA(crRNA)pairs targeting the rat Hao1and Ldha genes were initially screened ex vivo.In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes,primarily resulting in small deletions.This resulted in decreased transcription and translational expression of Hao1 and Ldha.Treatment significantly reduced urine oxalate levels,reduced kidney damage,and alleviated nephrocalcinosis in rats with PH1.No liver toxicity,ex-liver genome editing,or obvious offtarget effects were detected.We demonstrated the AAVAsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1,serving as a proof-ofconcept for the development of multiplex genome editingbased gene therapy.

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Natural variants and editing events provide insights into routes for spike architecture modification in common wheat

Spike architecture is an indicative trait of grain yield in common wheat(Triticum aestivum).A segregating population was generated for mapping genes contributing to spike morphometric traits by crossing the two common wheat cultivars'CItr 17600'with branching spikes and'Yangmai 18'with normal spikes.A major quantitative trait locus for spike length was mapped to the Q5A region of chromosome 5A.Yangmai18 carried a Q5Ab allele for short spikes,which harbored one SNP in the last intron,and a 1-bp InDel in the 720-bp fragment from the start codon,compared to Q5Aa in Chinese Spring.CItr 17600 harbored a q5Ab allele for long spikes,which has a 6-bp deletion compared to the reported q5Aa allele that was involved in the binding site of microRNA 172(miR172).This 6-bp deletion in immediately upstream of this binding site was involved in changes of four amino acids.The natural q5A allele appeared to be rare in common wheat but frequent in tetraploid T.turgidum accessions with branching spikes.The CRISPR/Cas9 technology was used to edit the upstream region involving in the miR172 binding site in Yangmai 18 and identified two independent editing events,one with a 1-bp insertion in Q5A and the other with a 2-bp deletion in Q5D,resulting in several shapes of spikes in the transgenic progeny.In addition to the effects of natural q5A allele and the edited Q5A genes,this study indicated the regeneratability and transformability of Yangmai 18 as an elite cultivar.Altogether,this study provides insight into future modification and engineering of spike architecture in common wheat.

In-Silico Identification and Differential Analysis of Mitochondrial RNA Editing Events in Helianthus Genotypes/Species and Powdery Mildew Infected Variants

Sunflower is one of the most used commercial oilseed crops and suffers due to Powdery mildew. RNA sequence alteration occurs due to RNA editing which is a post transcriptional modification. It causes a deviation from the genomic DNA sequence resulting in RNA-DNA differences. Accurate study of RNA editing events in diverse species is possible by NGS based methods. Here, we performed RNA sequencing of 12 leaf transcriptomes, which include three genotypes of Helianthus annuus (2023B, TX16R and ID25), H. debilis, H. niveus, and H. praecox along with their respective powdery mildew pathogen infected variants and systematically analysed the mitochondrial RNA editing events using computational reference-based mapping approach. We discovered 687 editing sites, 220 editing events in the protein-coding regions, among all species and genotypes considered in this study. These included “C to U” and “U to C” RNA editing events. On further analysis, we observed that these editing events include 14 different types of amino acid changes that involve the creation of two stop codon events. The conserved editing sites identified were 247 accounting for ~36% of all the editing sites identified. This study provides a detailed picture of the Helianthus species’ mitochondrial RNA editing status. We have identified and characterized for the first time, genotype-specific, species-specific, and stress-specific RNA editing events which may be useful as a potential source for stress-responsive studies in the future.

Multiplex genome editing targeting soybean with ultra-low anti-nutritive oligosaccharides

Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.