MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems

Abstract

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

“…The main reason for this inconsistency in results is that our previous conclusions were based on only one or two T1 plants, and that different promoters were used to drive Cas9. Consistent with this notion, in our previous report on sgRNA targeting the ABI1 gene, the investigation of eight T1 abi1 mutants and two T2 populations for off-target mutations generated similar results to that observed in the present study (Zhang et al, 2017b). It will be interesting to use our egg cell-specific promoter-controlled (EPC) CRISPR/Cas9 system to determine the specificity of GAI-sgRNA1 since its specificity score was 64 relative to the range of 0-100, much lower than that (94) of the sgR-ETC2 (Haeussler et al, 2016;Hsu et al, 2013).…”

“…Therefore, it is not strange for the high-frequency off-target 11 / 26 11 / 26 mutations in the CPC off-target site harboring a mismatch in the 8 th nt in the PAM-proximal position ( Figure 1). Similar to this study, our previous investigation involving sgRNA-ABI1 indicated that the off-target sites also harbor a mismatch in the 9 th nt in the PAM-proximal position (Zhang et al, 2017b). The frequency of off-target events was also affected by mismatch identity and could be largely indicated as: rN:dT ≥ rU:dG >> rC:dC >> rA/rG:dA/dG (Doench et al, 2016;Tsai and Joung, 2016;Tycko et al, 2016).…”

“…The results indicated that depending on Cas9 promoters, the frequencies of targeted T-DNA insertions were 6.7% (1/15), 83.3% (5/6), and 77.8% (7/9) for the three constructs, respectively ( Figure S3). In addition, we encountered the same problem for the analysis of mutations in the HAB1 gene of line #4 T1 and T2 plants in our previous study (Zhang et al, 2017b). This failure in PCR amplifications may also be due to T-DNA insertions.…”

“…S3). In addition, we encountered the same problem for the analysis of mutations in the HAB1 gene of line #4 T1 and T2 plants in our previous study (25). This failure in PCR amplifications may also be due to T-DNA insertions.…”

“…The 8 th and 9 th mismatches mentioned earlier were all of the rC:dT mismatch, suggesting that this mismatch identity was frequently tolerated and contributed to the off-target effects in plants. The observation that the off-target site harboring the two mismatches to the sgRNA targeting ABI1 displayed higher frequency off-target effects than that harboring one mismatch (Zhang et al, 2017b) indicates that more factors should be considered in the development of more precise off-target prediction algorithms that are based on large training data sets from high-throughput experiments. Because our EPC system is a relatively short-time expression system (Wang et al, 2015), it seemed that it should have a significantly lower off-target frequency than systems driven by constitutive promoters, including 2×35S, UBQ1, UBQ10, and PcUbi4-2 (Fauser et al, 2014;Feng et al, 2014;Peterson et al, 2016).…”