Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice

Abstract

The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0342-x) contains supplementary material, which is available to authorized users.

Biallelic Gene Targeting in Rice

“…Mutation frequency in calli cultured for one month seemed to be similar in gALS-2, -3, and -4 while that of gALS-1 seemed lower than the others (Supplemental Fig. S1B, Mikami et al, 2015a). To increase cleavage events at the targeted locus, we decided to use both gALS-2 and gALS-3 for DSB induction.…”

“…However, we did not have enough seeds of the genetically fixed Cas9 transgenic line because the majority of the transgenic lines were multicopy and transgenes were segregated in the progeny. Since we had already successfully established the sequential transformation system (Nishizawa-Yokoi et al, 2015b;Mikami et al, 2015aMikami et al, , 2015b, we decided to apply it to CRISPR/Cas9-mediated GT.…”

“…All-in-one vectors of Cas9, gALS-1 to -4 and HPT (pZH_OsU6gALS_MMCas9) were constructed according to the method described in Mikami et al (2015aMikami et al ( , 2015b. The binary vector pCAMBIA-sGFP (Toki 1997;Toki et al, 2006) was used as a control vector in GT experiments as it contains the HPT gene.…”

“…Especially for the positive-negative selection system used in rice GT (Terada et al, 2002(Terada et al, , 2007, efficient delivery of long and intact DNA is necessary, because positive and negative selection marker gene expression cassettes make the HDR template long. Using an established Agrobacterium-mediated transformation system (Toki et al, 2006), we succeeded in achieving CRISPR/Cas9-mediated targeted mutagenesis in rice (Endo et al, 2015;Mikami et al, 2015aMikami et al, , 2015b. Here, we report the establishment of an efficient break-induced GT system using Agrobacterium-mediated transformation to deliver both a repair construct and the CRISPR/Cas9 expression system.…”

Genome editing weds CRISPR: what is in it for phytoremediation?

“…T-DNA-delivered gRNA-Cas9 (in Agrobacterium mediated T-DNA transformation) has also been tested but due to transitory action of T-DNA in callus induction, activity has been observed in somatic tissues via genome integration. To make the most of this strategy, it might be imperative to amalgamate diverse gRNAs with Cas9 in a single T-DNA, as all-in-one plasmid approach would definitely improve editing (Bortesi & Fischer, 2015;Mikami, Toki & Endo, 2015). Cloning systems like Golden-Braid ease the association of pre-made DNA elements into multigene constructs (Vazquez-Vilar et al, 2015;Liu & Stewart, 2015).…”