Abstract
BackgroundCo-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.ResultsTo 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.ConclusionsThe 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.
Highlights
Co-expression of two distinct guide RNAs has been used to facilitate the application of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in fields such as large genomic deletion
Subsequent DNA sequencing results demonstrated that one (See figure on page.) Fig. 1 The design and stability of direct repeats (DRs)-involved paired-guide RNAs (gRNAs) plasmids pDG-A-X in E. coli. a The modular construction strategy of pDG-A-X series. pKB plasmid was used for Polymerase chain reaction (PCR) amplification of DNA part 1, which contained pDG-A-X backbone, one constitutive promoter J23119, and a gRNA scaffold. pKI plasmid was used for PCR amplification of part 2 series, which contained a gRNA fragment followed by another promoter J23119 and 20-bp space sequence
The results indicated that reversed paired-gRNA plasmids (RPGPs)-associated CRISPR/Cas9 system was successfully used for large genome editing in E. coli
Summary
Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas 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. The paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas system. CRISPR-based systems are powerful tools for genetic manipulations in both eukaryotic and prokaryotic organisms, which solely rely on single guide RNA molecule (gRNA) for targeting [1, 2]. When using CRISPR array, recombination between DRs of the array caused the loss of spacer sequences and the subsequent failure of genomic modification [2, 5]. Co-expressing multiple gRNAs in one plasmid resulted in self-homologous recombination if gRNA
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