The imperative aspect of the CRISPR/Cas9 system is a short stretch of 20 nucleotides of gRNA that control the overall specificity.Due to the small size,the chance of its multiple occurrences in the genome increases;how...The imperative aspect of the CRISPR/Cas9 system is a short stretch of 20 nucleotides of gRNA that control the overall specificity.Due to the small size,the chance of its multiple occurrences in the genome increases;however,a few mismatches are tolerated by the Cas9 endonuclease activity.An accurate and careful in silico-based off-target prediction while target selection is preferred to address the issue.These predictions are based on a comprehensive set of selectable parameters.Therefore,we investigated the possible off-target prediction and their screening in StERF3 gene-edited potato plants while developing StERF3-loss-of-function mutants using CRISPR/Cas9 approach.The 201 off-targets for the selected targets of the StERF3 gene were predicted,and 79 werefiltered as potential off-targets.Of these 79,twenty-five off-targets showed scores with defined cut-off values<0.5 and were analyzed in Sterf3-edited potato plants compared to wild-type plants.No off-targeting was found to have occurred in edited plants.展开更多
Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present...Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.展开更多
Cotton(Gossypium hirsutum L.)is one of the most important global crops that supports the textile industry and pro-vides a living for millions of farmers.The constantly increasing demand needs a significant rise in cot...Cotton(Gossypium hirsutum L.)is one of the most important global crops that supports the textile industry and pro-vides a living for millions of farmers.The constantly increasing demand needs a significant rise in cotton production.Genome editing technology,specifically with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)tools,has opened new possibilities for trait development in cotton.It allows pre-cise and efficient manipulation within the cotton genome when compared with other genetic engineering tools.Current developments in CRISPR/Cas technology,including prime editing,base editing,and multiplexing editing,have expanded the scope of traits in cotton breeding that can be targeted.CRISPR/Cas genome editing has been employed to generate effectively CRISPRized cotton plants with enhanced agronomic traits,including fiber yield and quality,oil improvement,stress resistance,and enhanced nutrition.Here we summarized the various target genes within the cotton genome which have been successfully altered with CRISPR/Cas tools.However,some challenges remain,cotton is tetraploid genome having redundant gene sets and homologs making challenges for genome edit-ing.To ensure specificity and avoiding off-target effects,we need to optimize various parameters such as target site,guide RNA design,and choosing right Cas variants.We outline the future prospects of CRISPR/Cas in cotton breeding,suggesting areas for further research and innovation.A combination of speed breeding and CRISPR/Cas might be useful for fastening trait development in cotton.The potentials to create customized cotton cultivars with enhanced traits to meet the higher demands for the agriculture and textile industry.展开更多
The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation ...The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.展开更多
CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of ...CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.展开更多
As versatile and robust genome editing tools,clustered regularly interspaced short palindromic repeats(CRISPR)technologies have been broadly used in basic research,biotechnology,and therapeutic development.Off-target ...As versatile and robust genome editing tools,clustered regularly interspaced short palindromic repeats(CRISPR)technologies have been broadly used in basic research,biotechnology,and therapeutic development.Off-target mutagenesis by CRISPR systems has been demonstrated,and various methods have been developed to markedly increase their specificity.In this review,we highlight the efforts of producing and modifying guide RNA(gRNA)to minimize off-target activities,including sequence and structure design,tuning expression and chemical modification.The modalities of gRNA engineering can be applied across CRISPR systems.In conjunction with CRISPR protein effectors,the engineered gRNA enables efficient and precise genome editing.展开更多
The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the o...The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding. A 20-bp g RNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site. The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using g RNAs truncated by 1, 2, 3 and 5 bp, respectively. These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting. PCR was performed for each off-target site. All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared. The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed. The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively;rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively;all were 0% at the Off-MSTN-2 locus;rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively. The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower. This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp g RNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting. This finding will provide more effective gene editing strategy for use of CRISPR/Cas9 technology.展开更多
Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human...Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.展开更多
The specificity of the double-stranded RNA(dsRNA) used in the RNA interference(RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending rese...The specificity of the double-stranded RNA(dsRNA) used in the RNA interference(RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending research topic.However, the off-target effects of the dsRNA in RNAi are a major concern. In this study, the ds Hvβ’COPI(coat protein complex I, β’ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle(Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects. The RNA-seq results revealed that 63 and 44 differentially expressed genes(DEGs) were upregulated and downregulated, respectively, in the ds Hvβ’COPI treated group as compared with the control. Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR(RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results. Further downstream analysis revealed that there were no genes homologous with Hvβ’COPI in H. vigintioctopunctata. Additionally,no genes with a >11 bp continuous match with ds Hvβ’COPI were found in the H. vigintioctopunctata transcriptome. Six genes(Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and Hv E3 ubiquitin) were selected to examine the offtarget activity of ds Hvβ’COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways.The severity of silencing these six off-target genes was evaluated by employing RNAi. The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality. The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.展开更多
Site-directed RNA editing(SDRE)is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing.The past few years have witnessed a surge of interest in SDRE,with SDRE tool...Site-directed RNA editing(SDRE)is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing.The past few years have witnessed a surge of interest in SDRE,with SDRE tools emerging at a breathtaking pace.However,off-target effects of SDRE remain a tough problem,which constitutes a major hurdle to their clinical applications.Here we discuss the diverse strategies for combating off-target editing,drawing lessons from the published studies as well as our ongoing research.Overall,SDRE is still at its infancy,with significant challenges and exciting opportunities ahead.展开更多
A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control.However, the off-target effects of RNAi pest control are still not fully understood. Here, we studied...A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control.However, the off-target effects of RNAi pest control are still not fully understood. Here, we studied the off-target effects of two insecticidal siRNAs in both target and non-target insects. The results revealed that off-target effects of insecticidal siRNAs occur widely in both target and non-target insects. We classified the expression-changed genes according to their homology to the siRNA-targeted gene, related KEGG pathways with the siRNA-targeted gene and continuous matches with siRNAs. Surprisingly, the unintended significant changes in gene expression levels did not strictly match with the number of contiguous nucleotides in the siRNAs. As expected, the expression of small portions of the homologous and KEGG-related genes were significantly changed. We calculated the Shannon entropy of the transcriptome profile of the insects after injecting them with insecticidal siRNAs. Though hundreds of genes were affected in their expression levels post siRNAtreatment, the Shannon entropy of the transcriptome remained unchanged, suggesting that the transcriptome expression was balanced. Our results provide evidence that siRNAs cross-reacted with individual genes in non-target species, but did not have significant effects on the integrity of the transcriptome profiles in either target or non-target species on a genomic scale. The metric we proposed can be used to estimate the off-target effects of insecticidal siRNAs, which might be useful for evaluating the safety of RNAi in pest control.展开更多
DNA is the blueprint of life,instructing the growth,development,and reproduction of an organism.Genome sequencing uncovers the codes of life,whereas genome editing could rewrite the codes,thus then leads to revolution...DNA is the blueprint of life,instructing the growth,development,and reproduction of an organism.Genome sequencing uncovers the codes of life,whereas genome editing could rewrite the codes,thus then leads to revolutionary advances in all aspects of life sciences,such as uncovering regulatory network of life,increasing the production of crops,producing new breeds of livestock,and curing genetic disorders(Liu,2017).展开更多
Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during devel...Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during development. Researchers can add moieties like peptide nucleic acid or methoxyethyl-modified ribose sugars to enhance specificity and reduce toxicity. Current research suggests that challenges such as nonspecific action, interference at various stages, adverse reactions, and nuclease degradation may soon be manageable with advanced technologies. ASOs show particular promise in treating rare conditions like Duchenne Muscular Dystrophy (DMD) and Timothy syndrome. Stereopure ASOs with repeated left-right patterns offer increased potency and half-life due to their resistance to nuclease activity and improved cellular uptake. This review explores how technological advancements can enhance the use of ASOs to manage various rare disease conditions effectively. Despite challenges in development and application, ASO therapy holds the potential to become a viable treatment option for a wide range of rare diseases. Advances in technology offer the possibility of increasing specificity and reducing toxicity, making ASO therapy a more effective and safe treatment option for patients with rare diseases.展开更多
The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy.While complex genome editing in the mouse since the 1990 s has been dominated by the use of em...The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy.While complex genome editing in the mouse since the 1990 s has been dominated by the use of embryonic stem（ES） cells,CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes.Yet,this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project.Here,we describe the key steps leading to successful generation of a double knockout（KO） mouse by simultaneously targeting two homolog genes,Tmem176 a and Tmem176 b,which are located in the same genomic locus.Additionally,we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain.Thus,presented as a detailed case study that should be helpful to the non-specialists,we focus on the genotyping strategy to anticipate the various possibilities.展开更多
Rice(Oryza sativa L.)is an important staple food crop worldwide due to its adaptability to different environmental conditions.Because of its great economic and social importance,there is a constant requirement for new...Rice(Oryza sativa L.)is an important staple food crop worldwide due to its adaptability to different environmental conditions.Because of its great economic and social importance,there is a constant requirement for new varieties with improved agronomic characteristics,such as tolerance to different biotic(such as bacterium,fungus,insect and virus)and abiotic stresses(such as salinity,drought and temperature),higher yield and better organoleptic and nutritional value.Among the new genome editing technologies,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)(CRISPR/Cas)system allows precise and specific edition in a targeted genome region.It is one of the most frequently used techniques for the study of the function of new genes and for the development of mutant lines with enhanced tolerance to biotic and abiotic stresses,herbicide resistance or improved yield.The wide varieties of applications for this technology include simple non-homologous end joining,homologous recombination,gene replacement,and base editing.In this review,we analyzed how some of these applications have been used in rice cultivars to obtain rice varieties better adapted to current environmental conditions and market requirements.展开更多
The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, method...The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular...The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.展开更多
In October 2020,Dr.Emmanuelle Charpentier and Dr.Jennifer Doudna won the Nobel Prize in Chemistry for their pioneering work in precise genome editing using the CRISPR technology.Although CRISPR technology has develope...In October 2020,Dr.Emmanuelle Charpentier and Dr.Jennifer Doudna won the Nobel Prize in Chemistry for their pioneering work in precise genome editing using the CRISPR technology.Although CRISPR technology has developed rapidly in the last decade,there are still many uncertainties before eventual use in clinical settings.In this mini review,we summarize the current efforts in addressing the limitations of CRISPR technology and future directions.展开更多
The leading-edge CRISPR/CRISPR-associated technology is revolutionizing biotechnologies through genome editing.To track on/off-target events with emerging new editing techniques,improved bioinformatic tools are indisp...The leading-edge CRISPR/CRISPR-associated technology is revolutionizing biotechnologies through genome editing.To track on/off-target events with emerging new editing techniques,improved bioinformatic tools are indispensable.Existing tools suffer from limitations in speed and scalability,especially with whole-genome sequencing(WGS)data analysis.To address these limitations,we have developed a comprehensive tool called CRISPR-detector,a web-based and locally deployable pipeline for genome editing sequence analysis.The core analysis module of CRISPR-detector is based on the Sentieon TNscope pipeline,with additional novel annotation and visualization modules designed to fit CRISPR applications.Co-analysis of the treated and control samples is performed to remove existing background variants prior to genome editing.CRISPR-detector offers optimized scalability,enabling WGS data analysis beyond Browser Extensible Data file-defined regions,with improved accuracy due to haplotype-based variant calling to handle sequencing errors.In addition,the tool also provides integrated structural variation calling and includes functional and clinical annotations of editing-induced mutations appreciated by users.These advantages facilitate rapid and efficient detection of mutations induced by genome editing events,especially for datasets generated from WGS.The web-based version of CRISPR-detector is available at https://db.cngb.org/crispr-detector,and the locally deployable version is available at https://github.com/hlcas/CRISPR-detector.展开更多
CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1,...CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.展开更多
文摘The imperative aspect of the CRISPR/Cas9 system is a short stretch of 20 nucleotides of gRNA that control the overall specificity.Due to the small size,the chance of its multiple occurrences in the genome increases;however,a few mismatches are tolerated by the Cas9 endonuclease activity.An accurate and careful in silico-based off-target prediction while target selection is preferred to address the issue.These predictions are based on a comprehensive set of selectable parameters.Therefore,we investigated the possible off-target prediction and their screening in StERF3 gene-edited potato plants while developing StERF3-loss-of-function mutants using CRISPR/Cas9 approach.The 201 off-targets for the selected targets of the StERF3 gene were predicted,and 79 werefiltered as potential off-targets.Of these 79,twenty-five off-targets showed scores with defined cut-off values<0.5 and were analyzed in Sterf3-edited potato plants compared to wild-type plants.No off-targeting was found to have occurred in edited plants.
基金supported by the National Natural Science Foundation of China(82404798)the Natural Science Foundation of Sichuan Province(2024NSFSC1831)+1 种基金the National Key Laboratory for Tropical Crop Breeding(NKLTCB-RC202403,NKLTCBZRJJ4)the Hainan Seed Industrial Laboratory(B22C1000P).
文摘Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.
文摘Cotton(Gossypium hirsutum L.)is one of the most important global crops that supports the textile industry and pro-vides a living for millions of farmers.The constantly increasing demand needs a significant rise in cotton production.Genome editing technology,specifically with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)tools,has opened new possibilities for trait development in cotton.It allows pre-cise and efficient manipulation within the cotton genome when compared with other genetic engineering tools.Current developments in CRISPR/Cas technology,including prime editing,base editing,and multiplexing editing,have expanded the scope of traits in cotton breeding that can be targeted.CRISPR/Cas genome editing has been employed to generate effectively CRISPRized cotton plants with enhanced agronomic traits,including fiber yield and quality,oil improvement,stress resistance,and enhanced nutrition.Here we summarized the various target genes within the cotton genome which have been successfully altered with CRISPR/Cas tools.However,some challenges remain,cotton is tetraploid genome having redundant gene sets and homologs making challenges for genome edit-ing.To ensure specificity and avoiding off-target effects,we need to optimize various parameters such as target site,guide RNA design,and choosing right Cas variants.We outline the future prospects of CRISPR/Cas in cotton breeding,suggesting areas for further research and innovation.A combination of speed breeding and CRISPR/Cas might be useful for fastening trait development in cotton.The potentials to create customized cotton cultivars with enhanced traits to meet the higher demands for the agriculture and textile industry.
基金supported by the National Key Research and Development Program of China(Grant No.2022YFD1201600)Earmarked Fund for China Agriculture Research System(Grant No.CARS-26)+1 种基金Chongqing Natural Science Foundation Project(Grant No.CSTB2023NSCQ-MSX1085)Cultivar Improvement of Nanfeng Orange.
文摘The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.
基金supported by the grants 81771230(W.C.),31922048(E.Z.)and 31522037(H.Y.)from the National Natural Science Foundation of China.
文摘CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.
基金supported by the National Key Research and Development Program of China(2018YFA0801401 and 2019YFA0802801)the National Natural Science Foundation of China 31871345+1 种基金the Young Thousand Talented Program from Wuhan Universitythe startup funding from Wuhan University to H.Y.
文摘As versatile and robust genome editing tools,clustered regularly interspaced short palindromic repeats(CRISPR)technologies have been broadly used in basic research,biotechnology,and therapeutic development.Off-target mutagenesis by CRISPR systems has been demonstrated,and various methods have been developed to markedly increase their specificity.In this review,we highlight the efforts of producing and modifying guide RNA(gRNA)to minimize off-target activities,including sequence and structure design,tuning expression and chemical modification.The modalities of gRNA engineering can be applied across CRISPR systems.In conjunction with CRISPR protein effectors,the engineered gRNA enables efficient and precise genome editing.
基金supported by the National Transgenic Project of China (2016ZX08010001-002 and 2016ZX08010005-001)the National Natural Science Foundation of China (81471001)the Inner Mongolia Science and Technology Program, China (201502073)
文摘The CRISPR/Cas9 mediates efficient gene editing but has off-target effects inconducive to animal breeding. In this study, the efficacy of CRISPR/Cas9 vectors containing different lengths of g RNA in reduction of the off-target phenomenon in the bovine MSTN gene knockout fibroblast cell lines was assessed, providing insight into improved methods for livestock breeding. A 20-bp g RNA was designed for the second exon of the bovine MSTN gene, and CRISPR/Cas9-B was constructed to guide the Cas9 protein to the AGAACCAGGAGAAGATGGACTGG site. The alternative CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 vectors were constructed using g RNAs truncated by 1, 2, 3 and 5 bp, respectively. These vectors were then introduced into bovine fetal fibroblasts by the electroporation method, and single cells were obtained by flow cytometry sorting. PCR was performed for each off-target site. All samples were sequenced and analyzed, and finally the efficiency of each vector in target and off-target sites was compared. The CRISPR/Cas9-B vector successfully knocked out the MSTN gene, but the off-target phenomenon was observed. The efficiencies of CRISPR/Cas-B, CRISPR/Cas9-19, CRISPR/Cas9-18, CRISPR/Cas9-17 and CRISPR/Cas9-15 in triggering gene mutations at MSTN targeting sites were 62.16, 17.39, 7.69, 74.29 and 3.85%, respectively;rates of each at the Off-MSTN-1 locus were 52.86, 0, 0, 8.82 and 0%, respectively;all were 0% at the Off-MSTN-2 locus;rates at the Off-MSTN-3 site were 44.87, 51.72, 86.36, 0 and 50%, respectively. The efficiency of the CRISPR/Cas9-17 plasmid in the MSTN site was higher than that in the CRISPR/Cas9-B plasmid, and the effect at the three off-target sites was significantly lower. This study demonstrated that the CRISPR/Cas9-17 plasmid constructed by truncating 3 bp g RNA can effectively reduce the off-target effect without reducing the efficiency of bovine MSTN gene targeting. This finding will provide more effective gene editing strategy for use of CRISPR/Cas9 technology.
基金partially supported by the National Natural Science Foundation of China (No. 31110103904)the National Program on Key Basic Research Project (973 Program) of the Ministry of Science and Technology of China (Nos. 2011CBA01000 and 2012CB945101)
文摘Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.
基金supported by the National Natural Science Foundation of China(31972269 and 32172500)the National Key R&D Program of China(2017YFD0200900)。
文摘The specificity of the double-stranded RNA(dsRNA) used in the RNA interference(RNAi) technique is crucial for the success of sequence-specific gene silencing. Currently, RNAi-mediated insect control is a trending research topic.However, the off-target effects of the dsRNA in RNAi are a major concern. In this study, the ds Hvβ’COPI(coat protein complex I, β’ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle(Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects. The RNA-seq results revealed that 63 and 44 differentially expressed genes(DEGs) were upregulated and downregulated, respectively, in the ds Hvβ’COPI treated group as compared with the control. Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR(RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results. Further downstream analysis revealed that there were no genes homologous with Hvβ’COPI in H. vigintioctopunctata. Additionally,no genes with a >11 bp continuous match with ds Hvβ’COPI were found in the H. vigintioctopunctata transcriptome. Six genes(Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and Hv E3 ubiquitin) were selected to examine the offtarget activity of ds Hvβ’COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways.The severity of silencing these six off-target genes was evaluated by employing RNAi. The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality. The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.
基金supported by the start-up package of the ShanghaiTech University.
文摘Site-directed RNA editing(SDRE)is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing.The past few years have witnessed a surge of interest in SDRE,with SDRE tools emerging at a breathtaking pace.However,off-target effects of SDRE remain a tough problem,which constitutes a major hurdle to their clinical applications.Here we discuss the diverse strategies for combating off-target editing,drawing lessons from the published studies as well as our ongoing research.Overall,SDRE is still at its infancy,with significant challenges and exciting opportunities ahead.
基金funded by grants from the National Science and Technology Major Project of China(2016ZX08011002)。
文摘A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control.However, the off-target effects of RNAi pest control are still not fully understood. Here, we studied the off-target effects of two insecticidal siRNAs in both target and non-target insects. The results revealed that off-target effects of insecticidal siRNAs occur widely in both target and non-target insects. We classified the expression-changed genes according to their homology to the siRNA-targeted gene, related KEGG pathways with the siRNA-targeted gene and continuous matches with siRNAs. Surprisingly, the unintended significant changes in gene expression levels did not strictly match with the number of contiguous nucleotides in the siRNAs. As expected, the expression of small portions of the homologous and KEGG-related genes were significantly changed. We calculated the Shannon entropy of the transcriptome profile of the insects after injecting them with insecticidal siRNAs. Though hundreds of genes were affected in their expression levels post siRNAtreatment, the Shannon entropy of the transcriptome remained unchanged, suggesting that the transcriptome expression was balanced. Our results provide evidence that siRNAs cross-reacted with individual genes in non-target species, but did not have significant effects on the integrity of the transcriptome profiles in either target or non-target species on a genomic scale. The metric we proposed can be used to estimate the off-target effects of insecticidal siRNAs, which might be useful for evaluating the safety of RNAi in pest control.
基金supported by the National Key R&D Program of China(2017YFC1001901)the National Natural Science Foundation(31971365 and 31601196)the Guangzhou Science and Technology Project(201803010020 and 201707010085).
文摘DNA is the blueprint of life,instructing the growth,development,and reproduction of an organism.Genome sequencing uncovers the codes of life,whereas genome editing could rewrite the codes,thus then leads to revolutionary advances in all aspects of life sciences,such as uncovering regulatory network of life,increasing the production of crops,producing new breeds of livestock,and curing genetic disorders(Liu,2017).
文摘Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during development. Researchers can add moieties like peptide nucleic acid or methoxyethyl-modified ribose sugars to enhance specificity and reduce toxicity. Current research suggests that challenges such as nonspecific action, interference at various stages, adverse reactions, and nuclease degradation may soon be manageable with advanced technologies. ASOs show particular promise in treating rare conditions like Duchenne Muscular Dystrophy (DMD) and Timothy syndrome. Stereopure ASOs with repeated left-right patterns offer increased potency and half-life due to their resistance to nuclease activity and improved cellular uptake. This review explores how technological advancements can enhance the use of ASOs to manage various rare disease conditions effectively. Despite challenges in development and application, ASO therapy holds the potential to become a viable treatment option for a wide range of rare diseases. Advances in technology offer the possibility of increasing specificity and reducing toxicity, making ASO therapy a more effective and safe treatment option for patients with rare diseases.
基金supported by the Labex IGO project(n°ANR11-LABX-0016-01)funded by the "Investissements d'Avenir" French Government program,managed by the French National Research Agency(ANR)+1 种基金the context of the IHU-Cesti project(EXT173947) which received French government financial support managed by the National Research Agency via the "Investment Into The Future program" ANR-10-IBHU-005supported by Nantes Metropole and Region Pays de la Loire. C.L.was supported by Fondation Progreffe
文摘The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy.While complex genome editing in the mouse since the 1990 s has been dominated by the use of embryonic stem（ES） cells,CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes.Yet,this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project.Here,we describe the key steps leading to successful generation of a double knockout（KO） mouse by simultaneously targeting two homolog genes,Tmem176 a and Tmem176 b,which are located in the same genomic locus.Additionally,we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain.Thus,presented as a detailed case study that should be helpful to the non-specialists,we focus on the genotyping strategy to anticipate the various possibilities.
文摘Rice(Oryza sativa L.)is an important staple food crop worldwide due to its adaptability to different environmental conditions.Because of its great economic and social importance,there is a constant requirement for new varieties with improved agronomic characteristics,such as tolerance to different biotic(such as bacterium,fungus,insect and virus)and abiotic stresses(such as salinity,drought and temperature),higher yield and better organoleptic and nutritional value.Among the new genome editing technologies,the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)(CRISPR/Cas)system allows precise and specific edition in a targeted genome region.It is one of the most frequently used techniques for the study of the function of new genes and for the development of mutant lines with enhanced tolerance to biotic and abiotic stresses,herbicide resistance or improved yield.The wide varieties of applications for this technology include simple non-homologous end joining,homologous recombination,gene replacement,and base editing.In this review,we analyzed how some of these applications have been used in rice cultivars to obtain rice varieties better adapted to current environmental conditions and market requirements.
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.
基金supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of the People’s Republic of China(No.2015BAI09B03)the National Basic Research Program(973 Program)(No.2013CB35102)+3 种基金the National Natural Science Foundation of China(Nos.31371496 and31301008)the Natural Science Foundation of Hubei Province(No.2013CFB031)Wuhan Youth Chenguang Program of Science and Technology(No.2014072704011259)Tsinghua-Peking Center for Life Sciences
文摘The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.
基金supported by the National Natural Science Foundation of China(81870219)。
文摘In October 2020,Dr.Emmanuelle Charpentier and Dr.Jennifer Doudna won the Nobel Prize in Chemistry for their pioneering work in precise genome editing using the CRISPR technology.Although CRISPR technology has developed rapidly in the last decade,there are still many uncertainties before eventual use in clinical settings.In this mini review,we summarize the current efforts in addressing the limitations of CRISPR technology and future directions.
基金supported by the Fundamental Research Funds for the Central Public Welfare Research Institutes(ZZ13-YQ-095 and ZZXT201708)the Start-up Research Fund from BNU-HKBU United International College(UICR0700053-23).
文摘The leading-edge CRISPR/CRISPR-associated technology is revolutionizing biotechnologies through genome editing.To track on/off-target events with emerging new editing techniques,improved bioinformatic tools are indispensable.Existing tools suffer from limitations in speed and scalability,especially with whole-genome sequencing(WGS)data analysis.To address these limitations,we have developed a comprehensive tool called CRISPR-detector,a web-based and locally deployable pipeline for genome editing sequence analysis.The core analysis module of CRISPR-detector is based on the Sentieon TNscope pipeline,with additional novel annotation and visualization modules designed to fit CRISPR applications.Co-analysis of the treated and control samples is performed to remove existing background variants prior to genome editing.CRISPR-detector offers optimized scalability,enabling WGS data analysis beyond Browser Extensible Data file-defined regions,with improved accuracy due to haplotype-based variant calling to handle sequencing errors.In addition,the tool also provides integrated structural variation calling and includes functional and clinical annotations of editing-induced mutations appreciated by users.These advantages facilitate rapid and efficient detection of mutations induced by genome editing events,especially for datasets generated from WGS.The web-based version of CRISPR-detector is available at https://db.cngb.org/crispr-detector,and the locally deployable version is available at https://github.com/hlcas/CRISPR-detector.
基金National Natural Science Foundation of China(Grant No.31571403)the Natural Science Foundation of Beijing Municipality(Grant No.2171001)
文摘CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.
