Abstract
Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.
Highlights
All sgRNAs contained a spacer to direct Cas9 to a target in the phytoene desaturate gene of Nicotiana benthamiana (NbPDS1) and constructs were delivered to protoplasts isolated from leaves of N. benthamiana
Following a quantitative assessment of the frequency of mutagenesis by Illumina sequencing, we found no significant differences in the number of sequencing reads with mutations at the target (Fig 1B), indicating that neither human codon optimisation, the shorter stems found in the original sgRNA, or the minimal terminator significantly impaired the efficiency of mutagenesis in plants
Components of bacterial CRISPR/Cas systems have been applied to a wide variety of model and economically-important plant species including dicotyledonous fruit crops such as strawberries [52], tomatoes [53] and oranges [54] as well as monocotyledonous grain crops such as barley [35], wheat [55] and maize [56]
Summary
Additional Cas proteins from other bacterial species have been adapted for genome engineering in eukaryotes and have been applied to plants These include Cas from the Staphylococcus aureus Type II CRISPR system (SaCas9) [17,18,19] and Cas12a (previously Cpf1) from the Type V CRISPR systems found in Francisella novicida, Acidaminococcus sp. A few recent studies have attempted to compare Cas and Cas12a nucleases in similar experimental conditions They have necessarily used different targets with each type of nuclease, which are known to exert significant influence on efficiency [33]. A number of different studies, including several in plants, have reported so-called "off-target" mutations at sites in the genome with less than perfect identity to the spacer [34,35,36]. We present an analysis of all potential targets as well as their off-targets in the coding exons of Arabidopsis and test our tools at a larger number of targets to identify factors that influence efficiency
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