Abstract
Genome-editing CRISPR/Cas9 technology has led to the development of artificial transcriptional repressors, also known as CRISPR interference (CRISPRi). The deactivated Cas9 (dCas9) protein guided by crRNA can specifically bind to target DNA sequences, including promoters and operators, without DNA cleavage. Protospacer adjacent motif (PAM) sequence dependence may be disadvantageous in the design of target-specific CRISPRi, as the PAM sequence is essential for DNA cleavage by the CRISPR/Cas9 system. We constructed a chromosomally integrated dCas9 system (ΔaraBAD:dcas9) in Escherichia coli under the control of the L-arabinose-inducible PBAD promoter. Plasmids carrying various crRNAs with target sequences specific for the gal promoter (−10 region), and the galETK structural genes in the gal operon, were transformed into dCas9-expressing E. coli. Cellular growth and/or galactose metabolic rates were monitored in the presence or absence of gratuitous L-arabinose. D-galactose consumption and cell growth rates were partially retarded by targeting transcriptional elongation but were fully inhibited by targeting transcriptional initiation. Moreover, RT-qPCR analysis showed that CRISPRi with several modified PAM sequences can repress the transcription of target DNAs. These results indicate that cellular metabolic rates and cell growth can be controlled by targeting structural genes or regulatory regions using CRISPRi; also, a loose PAM sequence dependence can expand the DNA targets of CRISPRi.
Highlights
The biological role of CRISPR has been identified as a prokaryotic adaptive immune system (Mojica et al, 2005; Pourcel et al, 2005)
CRISPR interference can regulate the expression of DNA targets by inhibiting the transcription process
While nonsense point mutation can affect cells permanently by deleting the function of the target gene, dCas9mediated gene expression control allows the selection of desired time points (Quebatte and Dehio, 2017)
Summary
The biological role of CRISPR has been identified as a prokaryotic adaptive immune system (Mojica et al, 2005; Pourcel et al, 2005). A specific protospacer adjacent motif (PAM) (∼3–5 bp) sequence must be located near the target sequence for the proper operation of the CRISPR/Cas system (Rath et al, 2015). The 5 -NGG sequence should be located right after target DNA sequences in the case of CRISPR/Cas (O’connell et al, 2014). The CRISPR/Cas nuclease has been widely used in gene knockout studies in eukaryotic cells by the highly specific digestion of target DNA sequences followed by nonhomologous end joining, resulting in the insertion or deletion of target genes (Zhang et al, 2014b). Sitespecific genome editing can be achieved by the introduction of single-stranded oligonucleotides and/or double-stranded DNA fragments flanking homologous regions into prokaryotic or eukaryotic cells, respectively, which can be negatively selected by the CRISPR/Cas nuclease (Knott and Doudna, 2018)
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