Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potent...Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potential tool for genome editing;however,its application in B.subtilis remains unexplored.In this study,two IscB and enhanced IscB(enIscB)-based genome editing systems,named pBsuIscB and pBsuenIscB were established in B.subtilis SCK6,and their deletion efficiencies ranging from 13.3%to 100%.Compared to the pBsuIscB system,the pBsuenIscB system showed higher deletion efficiency,inducing the deletion of a large genomic fragment with a singleωRNA.Additionally,the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B.subtilis SCK6 genome.Lastly,the pBsuenIscB system could efficiently conduct a second round of genome editing in B.subtilis SCK6.This study indicates that IscB can be used for genome editing in B.subtilis,enabling the efficient construction of engineered B.subtilis strains for large-scale biomolecule production.展开更多
CRISPR-Cas系统是广泛存在于细菌和古菌中的防御系统。基于该系统开发的基因组编辑工具已在大量物种中实现靶向编辑。目前应用最多的是CRISPR-Cas9和CRISPR-Cas12a基因组编辑工具,但它们的蛋白大小均超过1000个氨基酸,不利于递送。来自...CRISPR-Cas系统是广泛存在于细菌和古菌中的防御系统。基于该系统开发的基因组编辑工具已在大量物种中实现靶向编辑。目前应用最多的是CRISPR-Cas9和CRISPR-Cas12a基因组编辑工具,但它们的蛋白大小均超过1000个氨基酸,不利于递送。来自转座子家族的TnpB和IscB蛋白(大小约400个氨基酸)分别被认为是Cas12和Cas9的祖先蛋白,但其功能直到最近才被解析。它们被统称为专性移动元件引导活性(obligate mobile element-guided activity, OMEGA)蛋白,其引导RNA被称为ωRNA。此后,OMEGA系统成为了基因编辑领域的研究热点之一。OMEGA系统在三域生物中都有广泛分布,而且种类多样。对OMEGA系统的深入研究,将有助于开发精简、高效、安全的新型基因组编辑工具。本文围绕OMEGA系统的发现历程、结构特点、作用机制和在基因组编辑中的应用展开介绍,为新型基因组编辑工具的开发和优化提供参考。展开更多
In a very recent article published in Science,Altae-Tran et al.recon-structed the evolution of CRISPR-Cas9 systems and traced their ances-tors to unique groups of transposons(Altae-Tran et al.,2021).These transposable...In a very recent article published in Science,Altae-Tran et al.recon-structed the evolution of CRISPR-Cas9 systems and traced their ances-tors to unique groups of transposons(Altae-Tran et al.,2021).These transposable elements encode RNA-guided nucleases that show strong potential for developing novel biotechnologies.Structural domains of these nucleases serve as useful building blocks for engineering novel RNA-guided nucleases via synthetic biology to strongly inspire the de-velopment of novel and precision genome-editing tools.展开更多
基金supported by the National Natural Science Founda-tion of China(Number:32402894)the Sichuan Science and Technology Program(Number:2024NSFSC0373).
文摘Existing CRISPR-based genome editing techniques for Bacillus subtilis(B.subtilis)are limited due to the large size of the cas gene.IscB,a recently reported DNA nuclease,is one-third the size of Cas9,making it a potential tool for genome editing;however,its application in B.subtilis remains unexplored.In this study,two IscB and enhanced IscB(enIscB)-based genome editing systems,named pBsuIscB and pBsuenIscB were established in B.subtilis SCK6,and their deletion efficiencies ranging from 13.3%to 100%.Compared to the pBsuIscB system,the pBsuenIscB system showed higher deletion efficiency,inducing the deletion of a large genomic fragment with a singleωRNA.Additionally,the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B.subtilis SCK6 genome.Lastly,the pBsuenIscB system could efficiently conduct a second round of genome editing in B.subtilis SCK6.This study indicates that IscB can be used for genome editing in B.subtilis,enabling the efficient construction of engineered B.subtilis strains for large-scale biomolecule production.
文摘CRISPR-Cas系统是广泛存在于细菌和古菌中的防御系统。基于该系统开发的基因组编辑工具已在大量物种中实现靶向编辑。目前应用最多的是CRISPR-Cas9和CRISPR-Cas12a基因组编辑工具,但它们的蛋白大小均超过1000个氨基酸,不利于递送。来自转座子家族的TnpB和IscB蛋白(大小约400个氨基酸)分别被认为是Cas12和Cas9的祖先蛋白,但其功能直到最近才被解析。它们被统称为专性移动元件引导活性(obligate mobile element-guided activity, OMEGA)蛋白,其引导RNA被称为ωRNA。此后,OMEGA系统成为了基因编辑领域的研究热点之一。OMEGA系统在三域生物中都有广泛分布,而且种类多样。对OMEGA系统的深入研究,将有助于开发精简、高效、安全的新型基因组编辑工具。本文围绕OMEGA系统的发现历程、结构特点、作用机制和在基因组编辑中的应用展开介绍,为新型基因组编辑工具的开发和优化提供参考。
基金supported by grants from the National Key R&D Program of China(2020YFA0906800 to QS)the National Natural Science Foundation of China(Grant No.31771380 to QS)State Kay Laboratory of Microbial Technology at Shandong University.
文摘In a very recent article published in Science,Altae-Tran et al.recon-structed the evolution of CRISPR-Cas9 systems and traced their ances-tors to unique groups of transposons(Altae-Tran et al.,2021).These transposable elements encode RNA-guided nucleases that show strong potential for developing novel biotechnologies.Structural domains of these nucleases serve as useful building blocks for engineering novel RNA-guided nucleases via synthetic biology to strongly inspire the de-velopment of novel and precision genome-editing tools.
