Genome editing,particularly using the CRISPR/Cas system,has revolutionized biological research and crop improvement.Despite the widespread use of CRISPR/Cas9,it faces limitations such as PAM sequence requirements and ...Genome editing,particularly using the CRISPR/Cas system,has revolutionized biological research and crop improvement.Despite the widespread use of CRISPR/Cas9,it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells.The hypercompact Cas12f,derived from Acidibacillus sulfuroxidans(AsCas12f),stands out due to its small size of only 422 amino acids and its preference for a T-rich motif,presenting advantageous features over SpCas9.However,its editing efficiency is extremely low in plants.Recent studies have generated two AsCas12f variants,AsCas12f-YHAM and AsCas12f-HKRA,demonstrating higher editing efficiencies in mammalian cells,yet their performance in plants remains unexplored.In this study,through a systematic investigation of genome cleavage activity in rice,we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants,particularly for AsCas12f-HKRA,which achieved an editing efficiency of up to 53%.Furthermore,our analysis revealed that AsCas12f predominantly induces deletion in the target DNA,displaying a unique deletion pattern primarily concentrated at positions 12,13,23,and 24,resulting in deletion size mainly of 10 and 11 bp,suggesting significant potential for targeted DNA deletion using AsCas12f.These findings expand the toolbox for efficient genome editing in plants,offering promising prospects for precise genetic modifications in agriculture.展开更多
For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-b...For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture.Here,we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool.We systematically characterized AsCas12a,LbCas12a,LwaCas13a,and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection.Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input.Using this tool,we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field.Our method,from sample preparation to result readout,could be rapidly and easily deployed in the field.This system could be extended to other crop pathogens,including those that currently lack a detection method and have metabolite profiles that make detection challenging.This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping,transgene detection,and qualitative detection of gene expression in the field.展开更多
基金supported by the STI 2030—Major Projects(2023ZD04074)Guidance Foundation,the Sanya Institute of Nanjing Agricultural University(NAUSY-ZZ01)+3 种基金the Natural Science Foundation of Jiangsu(BK20210385,BK20212010)the Project of Zhongshan Biological Breeding Laboratory(ZSBBL-KY2023-04)the Jiangsu Province Key Research and Development Program(BE2023369)the Jiangsu“Innovative and Entrepreneurial Talent”program(to J.T.).
文摘Genome editing,particularly using the CRISPR/Cas system,has revolutionized biological research and crop improvement.Despite the widespread use of CRISPR/Cas9,it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells.The hypercompact Cas12f,derived from Acidibacillus sulfuroxidans(AsCas12f),stands out due to its small size of only 422 amino acids and its preference for a T-rich motif,presenting advantageous features over SpCas9.However,its editing efficiency is extremely low in plants.Recent studies have generated two AsCas12f variants,AsCas12f-YHAM and AsCas12f-HKRA,demonstrating higher editing efficiencies in mammalian cells,yet their performance in plants remains unexplored.In this study,through a systematic investigation of genome cleavage activity in rice,we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants,particularly for AsCas12f-HKRA,which achieved an editing efficiency of up to 53%.Furthermore,our analysis revealed that AsCas12f predominantly induces deletion in the target DNA,displaying a unique deletion pattern primarily concentrated at positions 12,13,23,and 24,resulting in deletion size mainly of 10 and 11 bp,suggesting significant potential for targeted DNA deletion using AsCas12f.These findings expand the toolbox for efficient genome editing in plants,offering promising prospects for precise genetic modifications in agriculture.
基金supported by the National Natural Science Foundation of China(31771808 and 32001551)the China Postdoctoral Science Foundation(2020M680779)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(S2021ZD03)。
文摘For some Cas nucleases,trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection.Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture.Here,we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool.We systematically characterized AsCas12a,LbCas12a,LwaCas13a,and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection.Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input.Using this tool,we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field.Our method,from sample preparation to result readout,could be rapidly and easily deployed in the field.This system could be extended to other crop pathogens,including those that currently lack a detection method and have metabolite profiles that make detection challenging.This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping,transgene detection,and qualitative detection of gene expression in the field.
