Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a target...Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.展开更多
The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control,thereby representing a major threat to human health.The de novo mutation of bacteria...The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control,thereby representing a major threat to human health.The de novo mutation of bacterial genomes is an essential mechanism by which bacteria acquire antibiotic resistance.Previously,deletion mutations within bacterial immune systems,ranging from dozens to thousands of base pairs(bps)in length,have been associated with the spread of antibiotic resistance.Most current methods for evaluating genomic structural variations(SVs)have concentrated on detecting them,rather than estimating the proportions of populations that carry distinct SVs.A better understanding of the distribution of mutations and subpopulations dynamics in bacterial populations is needed to appreciate antibiotic resistance evolution and movement of resistance genes through populations.Here,we propose a statistical model to estimate the proportions of genomic deletions in a mixed population based on Expectation–Maximization(EM)algorithms and next-generation sequencing(NGS)data.The method integrates both insert size and split-read mapping information to iteratively update estimated distributions.The proposed method was evaluated with three simulations that demonstrated the production of accurate estimations.The proposed method was then applied to investigate the horizontal transfers of antibiotic resistance genes in concert with changes in the CRISPR-Cas system of E.faecalis.展开更多
Infectious diseases are a serious threat to human health,and accurate,rapid and convenient early detection of pathogens is the first step of active treatment.Technologies that detect pathogens have advanced significan...Infectious diseases are a serious threat to human health,and accurate,rapid and convenient early detection of pathogens is the first step of active treatment.Technologies that detect pathogens have advanced significantly because of the development of fundamental disciplines and the integration of multidisciplinary fields.Among these technologies,nucleic acid detection technology is preferred because of its rapid measurement,accuracy and high sensitivity.The CRISPR/Cas system,consisting of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR‐associated(Cas),is an adaptive immune system that specifically recognizes,binds and cleaves exogenous invasive nucleic acids.The CRISPR/Cas system is widely found in bacteria and archaea.Researchers have developed nucleic acid detection technologies with single‐molecule sensitivity,single‐base precision specificity,portability and low cost based on the specific cleavage and trans‐cleavage activities of the CRISPR/Cas system.The next generation of in‐vitro diagnostics is shifting to nucleic acid technology because this technology shows promise in a wide range of applications in resource‐constrained environments.In this review,the development and mechanism of the CRISPR/Cas system are presented together with representative CRISPR/Cas applications in nucleic acid detection.Additionally,the review summarizes future perspectives and trends of the CRISPR/Cas system in nucleic acid detection.展开更多
Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype.Recent advances in clustered regularly interspaced short palindromic repeats(CRISPR)-Cas-mediated techn...Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype.Recent advances in clustered regularly interspaced short palindromic repeats(CRISPR)-Cas-mediated technolo-gies have provided an efficient tool for genetic engineering of cells and organisms.Here,we review the three emerging gene editing tools(ZFNs,TALENs,and CRISPR-Cas)and briefly introduce the principle,classification,and mechanisms of the CRISPR-Cas systems.Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems,as well as the novel base editor(BE),prime editor(PE),and CRISPR-associated transposase(CAST)technologies,are described in detail.In addition,we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications.Finally,the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.展开更多
In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,br...In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,breeding,and medical therapeutics.So far,four types of endonucleases have been adopted and optimized as gene-editing tools:meganuclease,ZFN,TALEN,and Cas nuclease from the CRISPR-Cas system.Each tool comes with its own advantages and limitations.Over the last ten years,RNA-guided Cas nucleases have been extensively investigated and successfully implemented in almost all mammalian cells due to their remarkable editing efficacy,high specificity,and flexibility in targeting the specific locus.Diverse Cas nuclease,together with meganuclease,ZFN,and TALEN,represent the key strategies for nuclease-based gene editing.However,systematic introductions and comparisons among four types of nucleases are not yet available.Here,we overview the capabilities of four types of nucleases along the development history of gene editing and describe the molecular mechanisms of substrate recognition and cleavage.Particularly,we summarize the promising CRISPR-Cas systems as well as modified tools applied for gene editing in the eukaryotic genome.Moreover,how the re-modulated nucleases and other nucleases,either naturally occurring or AI-designed,might manipulate DNA sequences is discussed and proposed.展开更多
CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized th...CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.展开更多
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)-bas...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.展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(CRISPR/Cas)system was discovered in bacteria and archaea as an adaptive immunity system to protect against exogenous DNA(...The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(CRISPR/Cas)system was discovered in bacteria and archaea as an adaptive immunity system to protect against exogenous DNA(Barrangou et al.,2007).This system has been developed into a powerful gene editing tool for eukaryotic genome manipulation that has therapeutic opportunities against hereditary diseases(Cong et al.,2013),especially monogenic diseases.展开更多
In a series of publications a special, tetraploid diplochromosomal division system to only two types of progeny cells (4n/4C/G1 and 2n/4C para-diploid) has been suggested to initiate preneoplasia that can lead to a ca...In a series of publications a special, tetraploid diplochromosomal division system to only two types of progeny cells (4n/4C/G1 and 2n/4C para-diploid) has been suggested to initiate preneoplasia that can lead to a cancerous pathway. Colorectal and other preneoplasia are known with the pathogenic, histological phases of hyperplasia to arrested adenoma/nevi that can give rise to dysplasia with high risk for cancer development. The present theme is to find solutions to tumorigenic unsolved, biological problems (queries), explainable from the tetraploid 4n-system, which would support its operation in the cancerous pathway. Presently admitted, the mutational sequencing of the cancer genome (cancer chemistry) cannot discover so-called “dark matter”, which herein is considered to be the queries. The solutions from the 4n-system were largely supported by mutated APC-induced same type of tetraploidy from the mitotic slippage process. But importantly, these behaviors and consequences could be linked to the beginning of hyperplastic lesions and their development to the arrest-phase of preneoplasia (polyps/nevi). Function of HFSMs is mostly unknown, but for Barrett’s esophagus, HFSMs (p53, p16ink4a) caused inactivation of the Rb gene, leading to dysplasia with 4n, aneuploid, abnormal cell cycles. In vitro models of the 4n-system from normal human cells recapitulated preneoplasia-like histopathological changes. It was speculated that the “cancer-crucial” step to dysplasia could be therapy-vulnerable to CRISPR-caspase editing, and perhaps antibody treatment. Additionally, the 4n-system with spontaneous cell-behaviors together with preneoplasia molecular data promises construction of a more truthful cancer-paradigm than from sequencing data alone.展开更多
CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to de...CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community.展开更多
Adva nces in genome editi ng tech no logy have revoluti on ized basic and applied biology research in recent years,particu?larly due to the newly emerged CRISPR/Cas technique(Ren et al.,2017b).The classical CRISPR/Cas...Adva nces in genome editi ng tech no logy have revoluti on ized basic and applied biology research in recent years,particu?larly due to the newly emerged CRISPR/Cas technique(Ren et al.,2017b).The classical CRISPR/Cas system was derived from a bacterial defense system,which consists of a single guide RNA(sgRNA)for precise targeting and one Cas protein for DNA binding and nuclease activity.Relying on high precision of CRISPR/Cas system,multiple functions have been exploited in addition to original nuclease activity,such as genome base editing,gene knock-down and activation,chromatin imaging systems etc.展开更多
Dear Editor,Clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is an adaptive immune system discovered in prokaryotic bacteria or archaea that can fend off invading nucleic acids.Be...Dear Editor,Clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is an adaptive immune system discovered in prokaryotic bacteria or archaea that can fend off invading nucleic acids.Because of its simplicity,high efficiency and versatility,CRISPR/Cas system-mediated genome editing has been widely applied in plant research and agricultural production.展开更多
The type I system is the most widely distributed CRISPR-Cas system identified so far.Recently,we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA-resembling RNA in...The type I system is the most widely distributed CRISPR-Cas system identified so far.Recently,we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA-resembling RNA in halophilic archaea.Here,we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I-B CRISPR system in Haloarcula hispanica.When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp,transformation efficiencies of the spacer-encoding plasmids were improved by over three orders of magnitude,indicating a significant loss of interference.However,by conducting whole-genome sequencing and measuring the growth curves of the hosts,we still detected DNA cleavage and its influence on cell growth.Intriguingly,when the spacer was shortened to 24 bp,the transcription of the target gene was downregulated to 10.80%,while both interference and primed adaptation disappeared.By modifying the lengths of the spacers,the expression of the target gene could be suppressed to varying degrees.Significantly,by designing crRNAs with different spacer lengths and targeting different genes,we achieved simultaneous gene editing(cdc6E)and gene regulation(crtB)for the first time with the endogenous type I CRISPR-Cas system.展开更多
Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants.Agrobacterium-mediated genetic transformation(AMGT)is the preferred method of CRIS...Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants.Agrobacterium-mediated genetic transformation(AMGT)is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process.In this article,we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents.We give an overview of the development of AMGT vectors including binary vector,superbinary vector,dual binary vector,and ternary vector systems.We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains.We focus in particular on the ternary vector system and the resources we developed.In summary,it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems,which are often integrated with morphogenic regulators.The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.展开更多
基金funded by the Genetically Modified Breeding Major Projects(no.2019ZX08010003-001-008 and no.2016ZX08010-002-008)the National Natural Science Foundation of China(no.U19A2022).
文摘Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.
基金This work was supported by the National Institutes of Health[Grant R15GM131390 to X.W.,Grant R01CA245294 to M.Z.,and Grant R01AI116610 to K.P.]。
文摘The emergence of antibiotic resistance in bacteria limits the availability of antibiotic choices for treatment and infection control,thereby representing a major threat to human health.The de novo mutation of bacterial genomes is an essential mechanism by which bacteria acquire antibiotic resistance.Previously,deletion mutations within bacterial immune systems,ranging from dozens to thousands of base pairs(bps)in length,have been associated with the spread of antibiotic resistance.Most current methods for evaluating genomic structural variations(SVs)have concentrated on detecting them,rather than estimating the proportions of populations that carry distinct SVs.A better understanding of the distribution of mutations and subpopulations dynamics in bacterial populations is needed to appreciate antibiotic resistance evolution and movement of resistance genes through populations.Here,we propose a statistical model to estimate the proportions of genomic deletions in a mixed population based on Expectation–Maximization(EM)algorithms and next-generation sequencing(NGS)data.The method integrates both insert size and split-read mapping information to iteratively update estimated distributions.The proposed method was evaluated with three simulations that demonstrated the production of accurate estimations.The proposed method was then applied to investigate the horizontal transfers of antibiotic resistance genes in concert with changes in the CRISPR-Cas system of E.faecalis.
基金Foundation of the Innovation Academy for Green Manufacture Institute,Chinese Academy of Sciences,Grant/Award Number:IAGM2020C31National Key Research and Development Program of China,Grant/Award Numbers:2019YFC1606600,2019YFC1606602。
文摘Infectious diseases are a serious threat to human health,and accurate,rapid and convenient early detection of pathogens is the first step of active treatment.Technologies that detect pathogens have advanced significantly because of the development of fundamental disciplines and the integration of multidisciplinary fields.Among these technologies,nucleic acid detection technology is preferred because of its rapid measurement,accuracy and high sensitivity.The CRISPR/Cas system,consisting of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR‐associated(Cas),is an adaptive immune system that specifically recognizes,binds and cleaves exogenous invasive nucleic acids.The CRISPR/Cas system is widely found in bacteria and archaea.Researchers have developed nucleic acid detection technologies with single‐molecule sensitivity,single‐base precision specificity,portability and low cost based on the specific cleavage and trans‐cleavage activities of the CRISPR/Cas system.The next generation of in‐vitro diagnostics is shifting to nucleic acid technology because this technology shows promise in a wide range of applications in resource‐constrained environments.In this review,the development and mechanism of the CRISPR/Cas system are presented together with representative CRISPR/Cas applications in nucleic acid detection.Additionally,the review summarizes future perspectives and trends of the CRISPR/Cas system in nucleic acid detection.
基金supported by National Natural Science Foundation of China(32170096)Fundamental Research Funds for the Central Universities(2662022SKPY001).
文摘Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype.Recent advances in clustered regularly interspaced short palindromic repeats(CRISPR)-Cas-mediated technolo-gies have provided an efficient tool for genetic engineering of cells and organisms.Here,we review the three emerging gene editing tools(ZFNs,TALENs,and CRISPR-Cas)and briefly introduce the principle,classification,and mechanisms of the CRISPR-Cas systems.Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems,as well as the novel base editor(BE),prime editor(PE),and CRISPR-associated transposase(CAST)technologies,are described in detail.In addition,we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications.Finally,the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.
基金supported by the Ministry of Agriculture and Rural Affairs of Chinathe National Natural Science Foundation of China(32150018)start-up funds from Tsinghua University,Beijing(J.J.G.L.)
文摘In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,breeding,and medical therapeutics.So far,four types of endonucleases have been adopted and optimized as gene-editing tools:meganuclease,ZFN,TALEN,and Cas nuclease from the CRISPR-Cas system.Each tool comes with its own advantages and limitations.Over the last ten years,RNA-guided Cas nucleases have been extensively investigated and successfully implemented in almost all mammalian cells due to their remarkable editing efficacy,high specificity,and flexibility in targeting the specific locus.Diverse Cas nuclease,together with meganuclease,ZFN,and TALEN,represent the key strategies for nuclease-based gene editing.However,systematic introductions and comparisons among four types of nucleases are not yet available.Here,we overview the capabilities of four types of nucleases along the development history of gene editing and describe the molecular mechanisms of substrate recognition and cleavage.Particularly,we summarize the promising CRISPR-Cas systems as well as modified tools applied for gene editing in the eukaryotic genome.Moreover,how the re-modulated nucleases and other nucleases,either naturally occurring or AI-designed,might manipulate DNA sequences is discussed and proposed.
基金funded by National Natural Science Foundation of China (No. 31901010)Jiangsu Specially Appointed Professorship Foundationthe Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine)。
文摘CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.
基金supported by the National Natural Science Foundation of China (82270355, 82270354, 81970134, 82030011, 31630093)the National Key Research and Development Program of China (2019YFA0801601, 2021YFA1101801)。
文摘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.
基金surported by Research Foundation of Shanghai General Hospital。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(CRISPR/Cas)system was discovered in bacteria and archaea as an adaptive immunity system to protect against exogenous DNA(Barrangou et al.,2007).This system has been developed into a powerful gene editing tool for eukaryotic genome manipulation that has therapeutic opportunities against hereditary diseases(Cong et al.,2013),especially monogenic diseases.
文摘In a series of publications a special, tetraploid diplochromosomal division system to only two types of progeny cells (4n/4C/G1 and 2n/4C para-diploid) has been suggested to initiate preneoplasia that can lead to a cancerous pathway. Colorectal and other preneoplasia are known with the pathogenic, histological phases of hyperplasia to arrested adenoma/nevi that can give rise to dysplasia with high risk for cancer development. The present theme is to find solutions to tumorigenic unsolved, biological problems (queries), explainable from the tetraploid 4n-system, which would support its operation in the cancerous pathway. Presently admitted, the mutational sequencing of the cancer genome (cancer chemistry) cannot discover so-called “dark matter”, which herein is considered to be the queries. The solutions from the 4n-system were largely supported by mutated APC-induced same type of tetraploidy from the mitotic slippage process. But importantly, these behaviors and consequences could be linked to the beginning of hyperplastic lesions and their development to the arrest-phase of preneoplasia (polyps/nevi). Function of HFSMs is mostly unknown, but for Barrett’s esophagus, HFSMs (p53, p16ink4a) caused inactivation of the Rb gene, leading to dysplasia with 4n, aneuploid, abnormal cell cycles. In vitro models of the 4n-system from normal human cells recapitulated preneoplasia-like histopathological changes. It was speculated that the “cancer-crucial” step to dysplasia could be therapy-vulnerable to CRISPR-caspase editing, and perhaps antibody treatment. Additionally, the 4n-system with spontaneous cell-behaviors together with preneoplasia molecular data promises construction of a more truthful cancer-paradigm than from sequencing data alone.
基金supported by the National Key Research and Development Program of China(award no.NK2022010204)to Y.Z.the National Natural Science Foundation of China(award nos.32270433,32101205,32072045,and 31960423)to X.T.,X.Z.,and Y.Z.+3 种基金the Sichuan Science and Technology Program(award no.2021JDRC0032)to Y.Z.the Technology Innovation and Application Development Program of Chongqing(award no.CSTC2021JSCX-CYLHX0001)to X.T.and Y.Z.supported by the National Science Foundation Plant Genome Research Program grant(award nos.IOS-1758745 and IOS2029889)USDA-AFRI Agricultural Innovations Through Gene Editing Program(award no.2021-67013-34554)to Y.Q.S.S.is a fellow of the Foundation for Food and Agriculture Research.
文摘CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community.
基金This work was supported by Genome Tagging Project and grants from the Chinese Academy of Sciences(XDB19010204)Shanghai Municipal Commission for Science and Technology(17411954900,17JC1400900,17JC1420102,16JC420500)the National Natural Science Foundation of China(3153004&81672117,31730062 and 31821004).
文摘Adva nces in genome editi ng tech no logy have revoluti on ized basic and applied biology research in recent years,particu?larly due to the newly emerged CRISPR/Cas technique(Ren et al.,2017b).The classical CRISPR/Cas system was derived from a bacterial defense system,which consists of a single guide RNA(sgRNA)for precise targeting and one Cas protein for DNA binding and nuclease activity.Relying on high precision of CRISPR/Cas system,multiple functions have been exploited in addition to original nuclease activity,such as genome base editing,gene knock-down and activation,chromatin imaging systems etc.
基金This work is funded by the National Natural Science Foundation of China(31972244 and 31930089).
文摘Dear Editor,Clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is an adaptive immune system discovered in prokaryotic bacteria or archaea that can fend off invading nucleic acids.Because of its simplicity,high efficiency and versatility,CRISPR/Cas system-mediated genome editing has been widely applied in plant research and agricultural production.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA24020101)National Key R&D Program of China(2020YFA0906800)National Natural Science Foundation of China(91751201,32150020,32100499).
文摘The type I system is the most widely distributed CRISPR-Cas system identified so far.Recently,we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA-resembling RNA in halophilic archaea.Here,we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I-B CRISPR system in Haloarcula hispanica.When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp,transformation efficiencies of the spacer-encoding plasmids were improved by over three orders of magnitude,indicating a significant loss of interference.However,by conducting whole-genome sequencing and measuring the growth curves of the hosts,we still detected DNA cleavage and its influence on cell growth.Intriguingly,when the spacer was shortened to 24 bp,the transcription of the target gene was downregulated to 10.80%,while both interference and primed adaptation disappeared.By modifying the lengths of the spacers,the expression of the target gene could be suppressed to varying degrees.Significantly,by designing crRNAs with different spacer lengths and targeting different genes,we achieved simultaneous gene editing(cdc6E)and gene regulation(crtB)for the first time with the endogenous type I CRISPR-Cas system.
基金Supported by grants from the National Crop Breeding Fund (2016YFD0101804)the National Natural Science Foundation of China (31872678 and 31670371)and the National Transgenic Research Project (2016ZX08009002).
文摘Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants.Agrobacterium-mediated genetic transformation(AMGT)is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process.In this article,we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents.We give an overview of the development of AMGT vectors including binary vector,superbinary vector,dual binary vector,and ternary vector systems.We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains.We focus in particular on the ternary vector system and the resources we developed.In summary,it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems,which are often integrated with morphogenic regulators.The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.
