Abstract:
BACKGROUND:Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. RESULTS:To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. CONCLUSIONS:The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.
journal_name
Microb Cell Factjournal_title
Microbial cell factoriesauthors
Ding T,Huang C,Liang Z,Ma X,Wang N,Huo YXdoi
10.1186/s12934-020-01321-4subject
Has Abstractpub_date
2020-03-10 00:00:00pages
63issue
1issn
1475-2859pii
10.1186/s12934-020-01321-4journal_volume
19pub_type
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
pub_type: 杂志文章
doi:10.1186/s12934-017-0666-0
更新日期:2017-03-27 00:00:00
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
pub_type: 杂志文章
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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journal_title:Microbial cell factories
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