As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controv...As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.展开更多
While CRISPR/Cas9-mediated genome editing technology has been experiencing a rapid transformation during the past few years,a recent report on NgAgo-mediated singlestranded DNA-guided genome editing may offer an attra...While CRISPR/Cas9-mediated genome editing technology has been experiencing a rapid transformation during the past few years,a recent report on NgAgo-mediated singlestranded DNA-guided genome editing may offer an attractive alternative for genome manipulation.While it’s too early to predict whether NgAgo will be able to compete with or be superior to CRISPR/Cas9,the scientific community is anxiously waiting for further optimization and broader applications of the NgAgo genome editing technology.展开更多
基金The datasets generated during the current study are available in the Sequence Read Archive(SRA)repository under the accession number PRJNA720376(BioProject)GenBank under the accession number PKKI00000000.
文摘As nucleic acid-guided endonucleases,some prokaryotic Argonautes have been used as programmable nucleases.Natronobacterium gregoryi Argonaute(NgAgo)has also been proposed for gene editing,but this remains very controversial.Until now,the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2(N.gregoryi sp2)have not been characterized.We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody.We also cultured the N.gregoryi sp2 strain and performed immunoprecipitation,chromatin immunoprecipitation(ChIP),and RNA immunoprecipitation(RIP)assays.The nucleic acids that endogenously bound NgAgo in N.gregoryi sp2 cells were sequenced and analyzed.The results showed that NgAgo endogenously bound RNA rather than DNA.NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA,transcriptional regulators,RNA polymerases,and RNA-binding proteins.NgAgo mainly binds to the transcripts inside genes or in their upstream sequences.Interestingly,the top enriched motif of peaks was the same as that of miR-1289,suggesting that NgAgo may regulate gene expression post-transcriptionally.GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes.These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.
基金The authors’laboratory research was supported in part by research grants from the National Institutes of Health(AR50142,AR054381,and AT004418 to RCH,HHL and TCH)the Scoliosis Research Society(to MJL),and the National Center for Advancing Translational Sciences(NCATS)of the National Institutes of Health(UL1 TR000430)All authors read the journal’s authorship agreement and the manuscript was reviewed and approved by all of the authors.
文摘While CRISPR/Cas9-mediated genome editing technology has been experiencing a rapid transformation during the past few years,a recent report on NgAgo-mediated singlestranded DNA-guided genome editing may offer an attractive alternative for genome manipulation.While it’s too early to predict whether NgAgo will be able to compete with or be superior to CRISPR/Cas9,the scientific community is anxiously waiting for further optimization and broader applications of the NgAgo genome editing technology.
