Abstract:
:Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 Cas9 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology-directed repair-mediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production, and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.
journal_name
Biotechnol Bioengjournal_title
Biotechnology and bioengineeringauthors
Yeo WL,Heng E,Tan LL,Lim YW,Lim YH,Hoon S,Zhao H,Zhang MM,Wong FTdoi
10.1002/bit.27021subject
Has Abstractpub_date
2019-09-01 00:00:00pages
2330-2338issue
9eissn
0006-3592issn
1097-0290journal_volume
116pub_type
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journal_title:Biotechnology and bioengineering
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journal_title:Biotechnology and bioengineering
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journal_title:Biotechnology and bioengineering
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journal_title:Biotechnology and bioengineering
pub_type: 杂志文章
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journal_title:Biotechnology and bioengineering
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