Gene Editing in Plants: Assessing the Variables Through a Simplified Case Study

Multiple variables that control the relative levels of successful heritable plant genome editing were addressed using simple case studies in Arabidopsis thaliana. The recent advent of genome editing technologies (especially CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) has revolutionized various fields of scientific research. The process is much more specific than previous mutagenic processes and allows for targeting of nearly any gene of interest for the creation of loss-of-function mutations and many other types of editing, including gene-replacement and gene activation. However, not all CRISPR construct designs are successful, due to several factors, including differences in the strength and cell- or tissue-type specificity of the regulatory elements used to express the Cas9 (CRISPR Associated protein 9) DNA nuclease and single guide RNA components, and differences in the relative editing efficiency at different target areas within a given gene. Here we compare the levels of editing created in Arabidopsis thaliana by CRISPR constructs containing either different promoters, or altered target sites with varied levels of guanine-cytosine base content. Additionally, nuclease activity at sites targeted by imperfectly matched single guide RNAs was observed, suggesting that while the primary goal of most CRISPR construct designs is to achieve rapid, robust, heritable gene editing, the formation of unintended mutations at other genomic loci must be carefully monitored.