Gene editing in plants: assessing the variables through a simplified case study

Abstract

Key message Multiple variables that control the relative levels of successful heritable plant genome editing were addressed using simple case studies in Arabidopsis thaliana. Abstract 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.Plant Molecular Biology (2020) 103:75-89Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

“…In higher plants, however, a canonical TATA-like box and well-conserved proximal USE are present in the U3 promoters 20,21 . Both of these promoters are capable of producing high levels of sgRNA, which typically have a length of~200 nucleotides 22 . In general, the Arabidopsis U6 (AtU6) and AtU3 promoters are extensively used in CRISPR/Cas9 vectors for genome editing in dicot plants, whereas the rice (Oryza sativa) U6 (OsU6) and OsU3 promoters are primarily applied in genome engineering in monocot plants 16,23 .…”

Functional Validation of cas9/guideRNA Constructs for Site-directed Mutagenesis of Triticale ABA8’OH1 loci

“…Several vector sets have been developed for genome editing in plants [27][28][29], [21], [30]. We have decided to use multiplasmid, flexible system developed by Čermák et al [21] available from plasmid repository ADDGENE, that enables targeted, specific modification of monocot genomes, because its utility has been demonstrated by several authors [11,12,31]. To verify whether multiple homeoalleles of the ABA8'OH-1 gene can be simultaneously addressed, we were taking into account the nature of individual target sites, gRNAs design, possibility of off-target activity and two different Cas9 variants.…”

“…Despite the great potential of the new biotechnological tools, not all cas9/gRNA construct designs are equally successful, which is due to several factors as reviewed by [9], [10] and [11]. Since the Cas9 mode of action, besides its requirement of a protospaceradjacent motif (PAM), relies on a specific interaction between gRNA and the target sequence, it is of great importance to implement particularly suitable sequences in cas9/gRNA constructs used for stable plant transformation [10].…”

Toward sustainable production of value‐added bioenergy and industrial oils in oilseed and biomass feedstocks

“…More careful selection of promoters to achieve the desired expression strength and timing of transgenes will also likely be needed. Moreover, using strategies such as artificial miRNAs and gene editing (e.g., clustered regularly interspaced short palindromic repeats) will allow selective suppression or knockout of non-desired pathways to tailor the metabolic background in the target oilseed or biomass crop for maximal unusual fatty acid production (Shockey, 2020).…”