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.

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.

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.

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.

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.

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 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.

Generation and characterization of a novel CYP3A1/2 double knockout rat model using CRISPR-Cas9 system

OBJECTIVE Cytochrome P450(CYP)3A accounts for nearly 30%of total CYP enzymes in human liver and participates in the metabolism of over 50%of clinical drugs.CYP3A also plays an important role in the chemical metabolism,toxicity,and carcinogenicity.New animal models are needed to investigate CYP3A functions.METHODS The CRISPR-Cas9 technology was used to generate Cyp3a1/2 double knockout rat model.The absence of Cyp3a1/2 expression was evaluated through PCR and immunostaining.Metabolic studies of the CYP3A substrates midazolam and nifedipine both in vitro and in vivo were conducted to verify that CYP3A1/2 was functional y inactive in KO rats.In addition,compensatory up-regulation of other P450 genes in Cyp3a1/2 KO rats was detected.RESULTS The Cyp3a1/2 double KO rats were viable and fertile,and had no obvious physiological abnormities.Compared with the wild-type(WT)rat,Cyp3a1/2 expression was completely absent in the liver of the KO rat.In vitro and in vivo metabolic studies of the CYP3A1/2 substrates indicated that CYP3A1/2 was functionally inactive in double KO rats.CONCLUSION The Cyp3a1/2 double KO rat model was successfully generated and characterized.The Cyp3a1/2 KO rats as a novel rodent animal model will be a valuable tool for the study of the physiological and pharmacological roles of CYP3A,and determining whether the absence of CYP3A results in non-CYP mediated metabolism or metabolism by other CYP isoforms.

Therapeutic gene editing strategies using CRISPR-Cas9 for theβ-hemoglobinopathies

With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

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.

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.

Development of herbicide resistance genes and their application in rice

Rice is one of the most important food crops in the world.Weeds seriously affect the rice yield and grain quality.In recent years,there are tremendous progresses in the research and application of herbicideresistant genes in rice worldwide.This article reviews the working mechanisms of six herbicides(glyphosate,glufosinate,acetolactate synthase inhibitor herbicides,acetyl-Co A carboxylase inhibitor herbicides,hydroxyhenylpyruvate dioxygenase(HPPD)inhibitor herbicides and dinitroaniline herbicides),the resistance mutations of the corresponding herbicide-target genes,and the herbicide detoxification mechanisms by non-target genes.Examples are provided on herbicide-resistant rice materials obtained by transformation of exogenous resistance genes,by artificial mutagenesis and mutant screening,and by modifying the target genes through gene editing.This paper also introduces the current application of herbicide-resistant rice,points out problems that may be caused by utilization of herbicide resistant rice and solutions to the problems,and discusses the future prospects for the development of herbicideresistant rice.

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.

Room for improvement in the treatment of pancreatic cancer: Novel opportunities from gene targeted therapy

Pancreatic cancer is one of the highest and in fact,unchanged mortality-associated tumor,with an exceptionally low survival rate due to its challenging diagnostic approach.So far,its treatment is based on a combination of approaches(such as surgical resection with or rarely without chemotherapeutic agents),but with finite limits.Thus,looking for additional space to improve pancreatic tumorigenesis therapeutic approach,research has focused on gene therapy with unexpectedly growing horizons not only for the treatment of inoperable pancreatic disease,but also for its early stages.In vivo gene delivery viral vectors,despite few disadvantages(possible immunogenicity,toxicity,mutagenicity,or high cost),could be one of the most efficient cancer gene therapeutic strategies for clinical application due to their superiority compared with other systems(ex vivo delivery strategies).Their dominance consists of simple preparation,easy operation and a wide range of functions.Adenoviruses are one of the most common used vectors,inducing strong immune as well as inflammatory reactions.Oncolytic virotherapy,using the above mentioned in vivo viral vectors,is one of the most promising nonpathogenic,highly-selective cytotoxic anti-cancer therapy using anti-cancer agents with high anti-tumor potency and strong oncolytic effect.There have been a variety of targeted therapeutic and pre-clinical strategies tested for gene therapy in pancreatic cancer such as gene-editing systems(e.g.,clustered regularly interspaced palindromic repeats-Cas9),RNA interference technology(e.g.,microRNAs,short hairpin RNA or small interfering RNA),adoptive immunotherapy and vaccination(e.g.,chimeric antigen receptor T-cell therapy)with encouraging results.

Combination of CRISPR/Cas9 System and CAR-T Cell Therapy:A New Era for Refractory and Relapsed Hematological Malignancies

Chimeric antigen receptor T(CAR-T)cell therapy is the novel treatment strategy for hematological malignancies such as acute lymphoblastic leukemia(ALL),lymphoma and multiple myeloma.However,treatment-related toxicities such as cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)have become significant hurdles to CAR-T treatment.Multiple strategies were established to alter the CAR structure on the genomic level to improve efficacy and reduce toxicities.Recently,the innovative gene-editing technology-clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease9(Cas9)system,which particularly exhibits preponderance in knock-in and knockout at specific sites,is widely utilized to manufacture CAR-T products.The application of CRISPR/Cas9 to CAR-T cell therapy has shown promising clinical results with minimal toxicity.In this review,we summarized the past achievements of CRISPR/Cas9 in CAR-T therapy and focused on the potential CAR-T targets.

Advances on genetic and genomic studies of ALV resistance

Avian leukosis(AL)is a general term for a variety of neoplastic diseases in avian caused by avian leukosis virus(ALV).No vaccine or drug is currently available for the disease.Therefore,the disease can result in severe economic losses in poultry flocks.Increasing the resistance of poultry to ALV may be one effective strategy.In this review,we provide an overview of the roles of genes associated with ALV infection in the poultry genome,including endogenous retroviruses,virus receptors,interferon-stimulated genes,and other immune-related genes.Furthermore,some methods and techniques that can improve ALV resistance in poultry are discussed.The objectives are willing to provide some valuable references for disease resistance breeding in poultry.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases

There are an estimated 10000 monogenic diseases affecting tens of millions of individuals worldwide.The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients.CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA.The complexity of genomic insults resulting in heritable disease requires patientspecific genome editing strategies with consideration of DNA repair pathways,and CRISPR/Cas systems of different types,species,and those with additional enzymatic capacity and/or delivery methods.In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair,non-homologous end joining,microhomology-mediated end joining,and base editing to permanently correct diverse monogenic diseases.

A cluster of mutagenesis revealed an osmotic regulatory role of the OsPIP1 genes in enhancing rice salt tolerance

Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.