Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexp...Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.展开更多
Background Toxoplasma gondii oocysts,excreted in cat feces,pose a significant health risk to humans through contaminated soil and water.Rapid and accurate detection of T.gondii in environmental samples is essential fo...Background Toxoplasma gondii oocysts,excreted in cat feces,pose a significant health risk to humans through contaminated soil and water.Rapid and accurate detection of T.gondii in environmental samples is essential for public health.Methods We developed a novel,single-tube detection method that integrates loop-mediated isothermal amplifiication(LAMP),the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12b system,and lateral flow immunoassay strips for rapid,visual identification of T.gondii.This method targeted the T.gondiiB1 gene,initially amplifies it with LAMP,directed by a single-guide RNA(sgRNA).It then recognizes the amplified target gene and activates trans-cleavage,cutting nearby single-stranded DNA(ssDNA)reporters.Fluorescence detection was performed using a 6-Carboxyfluorescein(FAM)-12N-BlackHoleQuencher-1(BHQ1)reporter,while.Fluorescein Isothiocyanate(FITC)-12N-Biotin enabled visual detection on lateral flow strips.The method was tested for its ability to detect various T.gondii genotypes and related parasites,assessing its specificity and broad-spectrum applicability.It was further applied to real-world environmental samples to evaluate its practicality.Results The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability,successfully identifying nine T.gondii genotypes and distinguishing them from 11 other parasitic species.Sensitivity testing at both molecular(plasmid)and practical(oocyst)levels showed detection limits of 10 copies/μL and 0.1 oocyst,respectively.When applied to 112 environmental samples(soil,water,and cat feces),the method demonstrated 100%sensitivity,accurately reflecting known infection rates.Conclusions This LAMP-CRISPR/Cas12b single-tube method offers a robust,innovative approach for monitoring zoonotic T.gondii in environmental samples,with significant implications for public health surveillance.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an N...Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.展开更多
基金supported by the National Key Research and Development Program of China(2021YFC2701103,2021YFA0910602,and 2019YFA0802804)the National Natural Science Foundation of China(82070258 and 31925011)+1 种基金Open Research Fund of State Key Laboratory of Genetic Engineering,Fudan University(SKLGE-2104)Science and Technology Research Program of Shanghai(19DZ2282100)。
文摘Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b(CRISPR-Cas12b)nucleases have been computationally identified,yet their potential for genome editing remains largely unexplored.In this study,we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing,identifying five active candidates.Candidatus hydrogenedentes Cas12b(ChCas12b)was found to recognize a straightforward WTN(W=T or A)proto-spacer adjacent motif(PAM),thereby dramatically expanding the targeting scope.Upon optimization of the single guide RNA(sgRNA)scaffold,ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci.Additionally,we identified nine mutations enhancing ChCas12b specificity.More importantly,we demonstrated that both ChCas12b and its high-fidelity variant,ChCas12b-D496A,enabled allelespecific disruption of genes harboring single nucleotide polymorphisms(SNPs).These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.
基金Project support is provided by the National Key Research and Develop‑ment Program of China(Grant Nos.2021YFC2300800,2021YFC2300802 and 2021YFC2300804)Shanxi Provincial Agricultural and Rural Research Program(Grant No.LXXMsxnd202101)+1 种基金the Research Fund of Shanxi Province for Intro‑duced High‑level Leading Talents(Grant No.RFSXIHLT202101)the Special Research Fund of Shanxi Agricultural University for High‑level Talents(Grant No.2021XG001).
文摘Background Toxoplasma gondii oocysts,excreted in cat feces,pose a significant health risk to humans through contaminated soil and water.Rapid and accurate detection of T.gondii in environmental samples is essential for public health.Methods We developed a novel,single-tube detection method that integrates loop-mediated isothermal amplifiication(LAMP),the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12b system,and lateral flow immunoassay strips for rapid,visual identification of T.gondii.This method targeted the T.gondiiB1 gene,initially amplifies it with LAMP,directed by a single-guide RNA(sgRNA).It then recognizes the amplified target gene and activates trans-cleavage,cutting nearby single-stranded DNA(ssDNA)reporters.Fluorescence detection was performed using a 6-Carboxyfluorescein(FAM)-12N-BlackHoleQuencher-1(BHQ1)reporter,while.Fluorescein Isothiocyanate(FITC)-12N-Biotin enabled visual detection on lateral flow strips.The method was tested for its ability to detect various T.gondii genotypes and related parasites,assessing its specificity and broad-spectrum applicability.It was further applied to real-world environmental samples to evaluate its practicality.Results The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability,successfully identifying nine T.gondii genotypes and distinguishing them from 11 other parasitic species.Sensitivity testing at both molecular(plasmid)and practical(oocyst)levels showed detection limits of 10 copies/μL and 0.1 oocyst,respectively.When applied to 112 environmental samples(soil,water,and cat feces),the method demonstrated 100%sensitivity,accurately reflecting known infection rates.Conclusions This LAMP-CRISPR/Cas12b single-tube method offers a robust,innovative approach for monitoring zoonotic T.gondii in environmental samples,with significant implications for public health surveillance.
基金Our plant genome editing research is supported by the National Science Foundation Plant Genome Research Program(IOS-1758745)USDA-NIFA Biotechnology Risk Assessment Research Program(2018-33522-28789)+1 种基金Foundation for Food and Agriculture Research(593603)Syngenta Biotechnology.
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.
