High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize

Abstract

BackgroundCRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice.ResultsWe optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize.ConclusionsThe examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0289-2) contains supplementary material, which is available to authorized users.

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

“…tRNAs represent a highly conserved class of RNA molecules which are recognized and precisely processed by RNase P and RNase Z 8 . Various tRNAs have been exploited to allow polycistronic gRNA production with high processing efficiencies 9,10 . tRNAs however, contain internal Pol-III promoters 8 .…”

OPAQUE11 Is a Central Hub of the Regulatory Network for Maize Endosperm Development and Nutrient Metabolism

“…We also produced GRMZM2G147685 knock-out mutant 163 alleles (five alleles, o11-cas9-1 to o11-cas9-5, Supplemental Figure 5A) using 164 the CRISPR-Cas9 system (Qi et al 2016a) and performed allelism tests by 165 crossing two independent knock-out alleles (o11-cas9-1 and o11-cas9-2) to was in the dicotyledon clade (Figure 5A and 186 Supplemental Dataset 1). Notably, O11 contained an extra 300 amino acids at 187 the N-terminus, which was found only in Sorghum bicolor and Setaria italic 188 (Supplemental Figure 6).…”

“…Agrobacterium tumefaciens strain EHA105 (Frame et al, 2002 For the allelism test, a CRISPR-Cas9 vector of GRMZM2G147685 was 707 constructed as described using a simplex editing strategy (Qi et al 2016a the Bradford assay (Bradford, 1976). Immunoblot analyses were performed as 737 described previously (Wang et al, 2014) using antibodies against the following nuclear proteins of 15 DAP maize immature kernels was carried out using a 754 previously described method (Qi et al, 2016b).…”

Towards CRISPR/Cas crops – bringing together genomics and genome editing

“…Concentration of free Cas9 was thus not a major limitation even when Cas9 was distributed among eight sgRNAs, which suggests that it is possible to target more sites simultaneously, although this has not been demonstrated. The tRNA‐based approach has also been successfully applied in maize (Qi et al ., ). Moreover, Tang et al .…”