Development and Therapeutic Applications of Precise Gene Editing Technology

The advent of gene editing represents one of the most transformative breakthroughs in life science,making genome manipulation more accessible than ever before.While traditional CRISPR/Cas-based gene editing,which involves double-strand DNA breaks(DSBs),excels at gene disruption,it is less effective for accurate gene modification.The limitation arises because DSBs are primarily repaired via non-homologous end joining(NHEJ),which tends to introduce indels at the break site.While homology directed repair(HDR)can achieve precise editing when a donor DNA template is provided,the reliance on DSBs often results in unintended genome damage.HDR is restricted to specific cell cycle phases,limiting its application.Currently,gene editing has evolved to unprecedented levels of precision without relying on DSB and HDR.The development of innovative systems,such as base editing,prime editing,and CRISPR-associated transposases(CASTs),now allow for precise editing ranging from single nucleotides to large DNA fragments.Base editors(BEs)enable the direct conversion of one nucleotide to another,and prime editors(PEs)further expand gene editing capabilities by allowing for the insertion,deletion,or alteration of small DNA fragments.The CAST system,a recent innovation,allows for the precise insertion of large DNA fragments at specific genomic locations.In recent years,the optimization of these precise gene editing tools has led to significant improvements in editing efficiency,specificity,and versatility,with advancements such as the creation of base editors for nucleotide transversions,enhanced prime editing systems for more efficient and precise modifications,and refined CAST systems for targeted large DNA insertions,expanding the range of applications for these tools.Concurrently,these advances are complemented by significant improvements in in vivo delivery methods,which have paved the way for therapeutic application of precise gene editing tools.Effective delivery systems are critical for the success of gene therapies,and recent developments in both viral and non-viral vectors have improved the efficiency and safety of gene editing.For instance,adeno-associated viruses(AAVs)are widely used due to their high transfection efficiency and low immunogenicity,though challenges such as limited cargo capacity and potential for immune responses remain.Non-viral delivery systems,including lipid nanoparticles(LNPs),offer an alternative with lower immunogenicity and higher payload capacity,although their transfection efficiency can be lower.The therapeutic potential of these precise gene editing technologies is vast,particularly in treating genetic disorders.Preclinical studies have demonstrated the effectiveness of base editing in correcting genetic mutations responsible for diseases such as cardiomyopathy,liver disease,and hereditary hearing loss.These technologies promise to treat symptoms and potentially cure the underlying genetic causes of these conditions.Meanwhile,challenges remain,such as optimizing the safety and specificity of gene editing tools,improving delivery systems,and overcoming off-target effects,all of which are critical for their successful application in clinical settings.In summary,the continuous evolution of precise gene editing technologies,combined with advancements in delivery systems,is driving the field toward new therapeutic applications that can potentially transform the treatment of genetic disorders by targeting their root causes.

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.

Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

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.

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.

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.

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.

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

Noncoding RNAs instruct the Cas9 nuclease to site speifillyl cleave DNA in the CRISPR/Cas9 system.Despite the high incidence of hepatocellular carcinoma(HCC),the patient's outcome is poor.As a result of the emergence of therapeutic resistance in HCC patients,dlinicians have faced difficulties in treating such tumor.In addition,CRISPR/Cas9 screens were used to identify genes that improve the dlinical response of HCC patients.It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer,with a particular emphasis on HCC as part of the current state of knowledge.Thus,in order to locate recent developments in oncology research,we examined both the Scopus database and the PubMed database.The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs.Drug resistance can be overcome with the help of the CRISPR/Cas9 system.HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening although this method is not without limitations.It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy.CRISPR and its applications to tumor research,particularly in HCC,are examined in this study through a review of the literature.

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.

Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

Manipulation of flowering time to develop cultivars with desired maturity dates is fundamental in plant breeding.It is desirable to generate polyploid rapeseed(Brassica napus L.)germplasm with varying flowering time controlled by a few genes.In the present study,Bna SVP,a rapeseed homolog of the Arabidopsis SVP(Short Vegetative Phase)gene,was characterized and a set of mutants was developed using a CRISPR/Cas9-based gene-editing tool.A single construct targeting multiple sites was successfully applied to precisely mutate four copies of Bna SVP.The induced mutations in these copies were stably transmitted to subsequent generations.Homozygous mutants with loss-of-function alleles and free transgenic elements were generated across the four Bna SVP homologs.All mutant T_(1)lines tested in two environments(summer and winter growing seasons)showed early-flowering phenotypes.The decrease in flowering time was correlated with the number of mutated Bna SVP alleles.The quadruple mutants showed the shortest flowering time,with a mean decrease of 40.6%–50.7%in length relative to the wild type under the two growth conditions.Our study demonstrates the quantitative involvement of Bna SVP copies in the regulation of flowering time and provides valuable resources for rapeseed breeding.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease

The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.

The CRIPSR/Cas gene-editing system——an immature but useful toolkit for experimental and clinical medicine

A Chinese scientist, Jiankui He, and his creation of the world ' s first genetically altered baby made headlines recently. As a newly developed gene-editing technique, the CRISPR/Cas system should not be applied to human beings for reproductive purposes until it has been extensively tested. However, numerous experimental research studies in human somatic, germline cells, and even in embryos, have been conducted, which have shown CRISPR/Cas to be a useful tool for human genome editing and a potential therapeutic method for future clinical use.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing

The typeⅡ prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox.However,its applications are still limited by its inefficient transduction.Herein,we present a novel gene vector,the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery.Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells.The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene,which was expected to inhibit the expression of PLK1.Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently.The transduction with ZEBRA was cell line dependent,which showed~10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones.Furthermore,ZEBRA induced highlevel expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene,and inhibited the tumor cell growth significantly.This zwitterionic polymerinspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.

Use of gene-editing technology to introduce targeted modifications in pigs

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

The concept of gene therapy for glaucoma:the dream that has not come true yet

Gene therapies,despite of being a relatively new therapeutic approach,have a potential to become an important alternative to current treatment strategies in glaucoma.Since glaucoma is not considered a single gene disease,the identified goals of gene therapy would be rather to provide neuroprotection of retinal ganglion cells,especially,in intraocular-pressure-independent manner.The most commonly reported type of vector for gene delivery in glaucoma studies is adeno-associated virus serotype 2 that has a high tro pism to retinal ganglion cells,res ulting in long-term expression and low immunogenic profile.The gene thera py studies recruit inducible and genetic animal models of optic neuropathy,like DBA/2J mice model of high-tension glaucoma and the optic nerve crush-model.Reported gene therapy-based neuroprotection of retinal ganglion cells is targeting specific genes translating to growth factors(i.e.,brain derived neurotrophic factor,and its receptor TrkB),regulation of apoptosis and neurodegeneration(i.e.,Bcl-xl,Xiap,FAS system,nicotinamide mononucleotide adenylyl transferase 2,Digit3 and Sarm1),immunomodulation(i.e.,Crry,C3 complement),modulation of neuroinflammation(i.e.,e rythropoietin),reduction of excitotoxicity(i.e.,Com KIlα)and transcription regulation(i.e.,Max,Nrf2).On the other hand,some of gene therapy studies focus on lowering intra ocular pressure,by impacting genes involved in both,decreasing aqueous humor production(i.e.,aquaporin 1),and increasing outflow facility(i.e.,COX2,prostaglandin F2a receptor,RhoA/RhoA kinase signaling pathway,MMP1,Myocilin).The goal of this review is to summarize the current stateof-art and the direction of development of gene therapy strategies for glaucomatous neuropathy.

Plastid RNA editing reduction accompanied with genetic variations in Cymbidium,a genus with diverse lifestyle modes

Recent sequencing efforts have broadly uncovered the evolutionary trajectory of plastid genomes(plastomes)of flowering plants in diverse habitats,yet our knowledge of the evolution of plastid posttranscriptional modifications is limited.In this study,we generated 11 complete plastomes and performed ultra-deep transcriptome sequencing to investigate the co-evolution of plastid RNA editing and genetic variation in Cymbidium,a genus with diverse trophic lifestyles.Genome size and gene content is reduced in terrestrial and green mycoheterotrophic orchids relative to their epiphytic relatives.This could be partly due to extensive losses and pseudogenization of ndh genes for the plastid NADH dehydrogenase-like complex,but independent pseudogenization of ndh genes has also occurred in the epiphyte C.mannii,which was reported to use strong crassulacean acid metabolism photosynthesis.RNA editing sites are abundant but variable in number among Cymbidium plastomes.The nearly twofold variation in editing abundance is mainly due to extensive reduction of ancestral editing sites in ndh transcripts of terrestrial,mycoheterotrophic,and C.mannii plastomes.The co-occurrence of editing reduction and pseudogenization in ndh genes suggests functional constraints on editing machinery may be relaxed,leading to nonrandom loss of ancestral edited sites via reduced editing efficiency.This study represents the first systematic examination of RNA editing evolution linked to plastid genome variation in a single genus.We also propose an explanation for how genomic and posttranscriptional variations might be affected by lifestyle-associated ecological adaptation strategies in Cymbidium.

Harnessing CRISPR-Cas system diversity for gene editing technologies

The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

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.