Abstract
Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants.
Highlights
Many gene families that regulate key processes are highly redundant and spread across diverse chromosomal locations in plants
For each of the seven genes we selected two guide RNAs (gRNAs) targets sites per gene using standard Cas9 targeting requirements. gRNA units, each consisting of a gRNA and Arabidopsis U6 promoter, were synthesized in clusters of four, and stacked together to generate a vector harboring a total of fourteen gRNA units
This contrasts with transcription activator-like effector nuclease (TALEN) and designer transcription activatorlike effector systems which have been shown to result in off-target editing in plants at low rates.[21,22,23]
Summary
Many gene families that regulate key processes are highly redundant and spread across diverse chromosomal locations in plants. To understand gene function this necessitates the ability to simultaneously target and mutate distinct loci in a highly specific manner without affecting other genes. The CRISPR/Cas system has emerged as a powerful tool to create targeted mutations in plants [1]. By co-expressing multiple guide RNAs (gRNAs) with the Cas enzyme in plants efficient multiplex mutation of gene families can be achieved. Current data suggests that off-target mutagenesis rates in plants are low [3, 4] or undetectable, this assessment is based on data from small scale targeting events. In many non-plant systems the use of Cas in targeted mutagenesis is off-set by considerable rates of off-target events.
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