Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

Abstract

SummaryGenome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.

CRISPR/Cas9-Mediated Generation of Pathogen-Resistant Tomato against Tomato Yellow Leaf Curl Virus and Powdery Mildew

“…Secondly, chromatin confirmation may significantly affect the accessibility of target sites and not be considered in sgRNA designing tools [40]. Thirdly, a recent report indicated Cas9 availability as one of the limiting factors in multiplexing studies [41]. Several sgRNAs were expressed simultaneously in the present work, and thus it is possible that gRNA activities were compromised due to increased competition for SpCas9 binding.…”

Disentangling cause and consequence: Genetic dissection of theDANGEROUS MIX2risk locus, and activation of the DM2h NLR in autoimmunity

“…; Gantner et al, 2019), using previously described target sites / sgRNAs for NRG1 editing (Qi et al, 2018). Different versions of pDGE vectors (Ordon et al, 2017; Ordon et al, 2019; Barthel et al, 2020) were used to generate dm2 and Nbnrg1 mutant lines by Sp Cas9. Respective target sites are provided in Figures S2 (Arabidopsis lines) and S3 ( Nbnrg1 ).…”

Differential requirement for the EDS1 catalytic triad inA. thalianaandN. benthamiana

“…All authors approved the final version. (a) Scheme of pDGE492 used for generation of Nb bak1 mutant plants; based on pDGE463 (Stuttmann et al, 2021). Guide RNAs were expressed under control of a tomato U3 promoter fragment (Stuttmann et al, 2021).…”

“…(a) Scheme of pDGE492 used for generation of Nb bak1 mutant plants; based on pDGE463 (Stuttmann et al, 2021). Guide RNAs were expressed under control of a tomato U3 promoter fragment (Stuttmann et al, 2021). Target sites are depicted, color code corresponds to panel b.…”