Targeted mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family suggests competition of multiplexed sgRNAs for Cas9 apoprotein

Abstract

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas9 machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal lab settings, such as in the classroom or citizen science projects. Spacer sequences targeting genes of interest can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic as well as transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family, we found that Cas9 expression from an EGG CELL1 (EC1) promoter resulted in tenfold lower mutation rates than expression from a UBIQUITIN10 (UBQ10) promoter. A collection of bona fide null mutations in all nine GRF genes could be established with little effort. Finally, we explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. Multiplexing caused strong interference effects: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species. Our results suggest potential detrimental genetic interaction between induced mutations as well as competition of CRISPR RNAs for a limiting amount of Cas9 apoprotein.