Development of a mono-promoter-driven CRISPR/Cas9 system in mammalian cells

Abstract

The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.

CRISPR/Cas9-Based Engineering of the Epigenome

“…This strategy can be combined with single-vector systems validated to express multiple gRNAs driven by polymerase-III promoters (Sakuma et al, 2014; Tsai et al, 2014), which may be useful to increase the effectiveness and flexibility of the gRNAs. Another noteworthy variation is to express gRNAs using polymerase-II promoters, a system that has proven highly efficient and adjustable for inducible expression (Nissim et al, 2014; Yoshioka et al, 2015). …”

Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing

“…These small nuclear RNA promoters are constitutively transcribed by RNA polymerase III (Pol III) and require a specific 5′ nucleotide to initiate transcription (5′-Guanine for U6 or 5′-Adenine for U3) (Jiang et al, 2013; Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013; Lowder et al, 2015). U6 and U3 promoters work well to express gRNAs in plants but they are not ideal for certain gene targeting applications due to lack of spatiotemporal control and the requirement of extra nucleotide restrictions on 5′ ends of target sequences or mismatch nucleotide additions on gRNA sequences (Gao and Zhao, 2014; Xie et al, 2015; Yoshioka et al, 2015; Tang et al, 2016). …”

“…An earlier study utilized this expression strategy for genome editing in wheat cells but obtained low Cas9 mutagenesis activity, presumably due to low activity of unprocessed Pol II expressed gRNAs (Upadhyay et al, 2013). Proper processing of Pol II expressed primary RNA for mature gRNAs has been demonstrated by utilizing the hammerhead and HDV ribozyme RNA self-cleavage system in yeast (Gao and Zhao, 2014), Arabidopsis (Gao, Y. et al, 2015), and mammalian cells (Yoshioka et al, 2015) (Figure 1B, top panel). Alternatively, the Pol II expressed gRNA containing primary RNA can be processed by the Csy4 RNA cleavage system as demonstrated in mammalian cells (Nissim et al, 2014) (Figure 1B, bottom panel).…”

“…Recently, Yoshioka and colleagues developed a mono-promoter CRISPR-Cas9 system in which both Cas9 and gRNA are expressed as a single transcript by a single Pol II promoter (Yoshioka et al, 2015). In their design, they put an HH-gRNA-HDV cassette ahead of Cas9 by linking two components with an internal ribosome entry site (IRES).…”

Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

“…Moreover, multiple gRNAs can be produced from a single promoter so that each gRNA can have similar concentrations. Indeed, it has been reported that multiple gRNAs are successfully produced from RNA Pol II promoters using the ribozyme strategy in rice and in animal cells (Yoshioka et al, 2015; Tang et al, 2016). Furthermore, CRISPR-Cpf1 DNA-editing in rice has been successfully performed by utilizing gRNA produced from our RGR system, probably because our RGR system produces gRNA molecules with precise ends (Tang et al, 2017).…”