Abstract
BackgroundOur knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background. To achieve a systematic understanding of such GxG interactions, it is desirable to develop genome editing tools that can be rapidly deployed across many different genetic varieties.ResultsWe present an efficient CRISPR/Cas9 toolbox of super module (SM) vectors. These vectors are based on a previously described fluorescence protein marker expressed in seeds allowing identification of transgene-free mutants. We have used this vector series to delete genomic regions ranging from 1.7 to 13 kb in different natural accessions of the wild plant Arabidopsis thaliana. Based on results from 53 pairs of sgRNAs targeting individual nucleotide binding site leucine-rich repeat (NLR) genes, we provide a comprehensive overview of obtaining heritable deletions.ConclusionsThe SM series of CRISPR/Cas9 vectors enables the rapid generation of transgene-free, genome edited plants for a diversity of functional studies.
Highlights
Our knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background
CRISPR/Cas9 super module (SM)‐gRNA‐shuffle‐in vector toolbox In plants, CRISPR/Cas9 components are typically introduced as transgenes
In summary, we have set up an efficient toolbox that facilitates knocking out genes or deleting DNA fragments in A. thaliana
Summary
Our knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background. An alternative approach to connecting genotype and phenotype is to exploit intraspecific variation, which has added the benefit of informing about genes and alleles that help organisms to adapt to their environment. This approach has in the past decade been fueled by the rapidly increasing knowledge of genetic variation in hundreds, if not thousands of individuals. That requires the reference strain to have a functional
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