CRISPR (clustered regularly interspaced short palindromic repeats)‐Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been d...CRISPR (clustered regularly interspaced short palindromic repeats)‐Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene‐editing tools. As the natural inhibitors of CRISPR‐Cas systems, anti‐CRISPRs (Acrs) can be used as the “off‐switch” for CRISPR‐Cas systems to limit the off‐target effects caused by Cas9. Since the discovery of CRISPR‐Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.展开更多
In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively ...In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.展开更多
Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system.Its incomplete oxidation system has many...Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system.Its incomplete oxidation system has many crucial industrial applications.However,it also leads to slow growth and low biomass,requiring further metabolic modification for balancing the cell growth and incomplete oxidation process.As a non-model strain,G.oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation.In the last 15 years,our laboratory attempted to deploy multiple CRISPR/Cas systems in different G.oxydans strains and found none of them as functional.In this study,Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1,while Cpf1–FokI was deployed to study its nuclease activity.A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G.oxydans but lacked the nuclease cleavage activity.Therefore,the CRISPR/Cpf1–FokI system based on FokI nuclease was constructed.Single-gene knockout with efficiency up to 100%and double-gene iterative editing were achieved in G.oxydans.Using this system,AcrVA6,the anti-CRISPR protein of G.oxydans was discovered for the first time,and efficient genome editing was realized.展开更多
CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Cren...CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum,usually contain both type I and typeⅢCRISPR-Cas systems.Two species,Saccharolobus solfataricus and Sulfolobus islandicus,have been important models for CRISPR study in archaea,and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps:adaptation,expression and crRNA processing,and interference.Four subtypes of CRISPR-Cas systems are common in these organisms,including I-A,I-D,Ⅲ-B,andⅢ-D.These cas genes form functional modules,e.g.,all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and i Cas modules.Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea,and these methodologies are useful in demonstration of the protospacer-adjacent motif(PAM)-dependent DNA interference by I-A interference modules and multiple interference activities byⅢ-B Cmr systems.Ribonucleoprotein effector complexes have been isolated for SulfolobalesⅢ-B andⅢ-D systems,and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.展开更多
基金Strategic Priority Research Programs of the Chinese Academy of Sciences,Grant/Award Number:XDA19050301National Natural Science Foundation of China,Grant/Award Number:31601189,81572433 and 81772646+1 种基金Biological Resources Program from Chinese Academy of Sciencesthe Young Elite Scientist Sponsorship Program by CAST,Grant/Award Number:2018QNRC001
文摘CRISPR (clustered regularly interspaced short palindromic repeats)‐Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene‐editing tools. As the natural inhibitors of CRISPR‐Cas systems, anti‐CRISPRs (Acrs) can be used as the “off‐switch” for CRISPR‐Cas systems to limit the off‐target effects caused by Cas9. Since the discovery of CRISPR‐Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.
基金supported by the National Key Research and Development Program of China(2023YFF1000200)the National Natural Science Foundation of China(U21A20276)+1 种基金the Tencent Foundation through the XPLORER PRIZE and the New Cornerstone Science Foundation to Z.H.,Heilongjiang Touyan Team(HITTY-20190034 to Z.H.)Natural Science Foundation of Heilongjiang Province of China(YQ2023C032 to Y.Z.)。
文摘In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.
基金This work was supported by the National Natural Science Foundation of China(Key Program,31830068),China National Postdoctoral Program for Innovative Talents(BX20230147),China Postdoctoral Science Foundation(2023M741406),Jiangsu Funding Program for Excellent Postdoctoral Talent(2023ZB631).
文摘Gluconobacter oxydans is an important Gram-negative industrial microorganism that produces vitamin C and other products due to its efficient membrane-bound dehydrogenase system.Its incomplete oxidation system has many crucial industrial applications.However,it also leads to slow growth and low biomass,requiring further metabolic modification for balancing the cell growth and incomplete oxidation process.As a non-model strain,G.oxydans lacks efficient genome editing tools and cannot perform rapid multi-gene editing and complex metabolic network regulation.In the last 15 years,our laboratory attempted to deploy multiple CRISPR/Cas systems in different G.oxydans strains and found none of them as functional.In this study,Cpf1-based or dCpf1-based CRISPRi was constructed to explore the targeted binding ability of Cpf1,while Cpf1–FokI was deployed to study its nuclease activity.A study on Cpf1 found that the CRISPR/Cpf1 system could locate the target genes in G.oxydans but lacked the nuclease cleavage activity.Therefore,the CRISPR/Cpf1–FokI system based on FokI nuclease was constructed.Single-gene knockout with efficiency up to 100%and double-gene iterative editing were achieved in G.oxydans.Using this system,AcrVA6,the anti-CRISPR protein of G.oxydans was discovered for the first time,and efficient genome editing was realized.
基金grants from the Chinese National Transgenic Science and Technology Program(2019ZX08010003 to QS)the National Natural Science Foundation of China(31771380 to QS)+1 种基金the Qingdao Applied Research Fund for postdoctoral researchers(62450079311107 to ZY)the State Key Laboratory of Microbial Technology and Shandong University。
文摘CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria.Organisms of Sulfolobales,an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum,usually contain both type I and typeⅢCRISPR-Cas systems.Two species,Saccharolobus solfataricus and Sulfolobus islandicus,have been important models for CRISPR study in archaea,and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps:adaptation,expression and crRNA processing,and interference.Four subtypes of CRISPR-Cas systems are common in these organisms,including I-A,I-D,Ⅲ-B,andⅢ-D.These cas genes form functional modules,e.g.,all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and i Cas modules.Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea,and these methodologies are useful in demonstration of the protospacer-adjacent motif(PAM)-dependent DNA interference by I-A interference modules and multiple interference activities byⅢ-B Cmr systems.Ribonucleoprotein effector complexes have been isolated for SulfolobalesⅢ-B andⅢ-D systems,and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.
