Abstract
RNA-guided endonucleases (RGENs) have been used for genome editing in various organisms. Here, we demonstrate a simple method for performing targeted mutagenesis and genotyping in a model moss species, Physcomitrella patens, using RGENs. We also performed targeted mutagenesis in a non-model moss, Scopelophilla cataractae, using a similar method (Nomura et al., 2016), indicating that this experimental system could be applied to a wide range of mosses species.
Keywords: Genome editing, RNA-guided endonucleases, CRISPR/Cas system, Targeted mutagenesis, Mosses
Background
Targeted mutagenesis using RNA-guided endonucleases (RGENs) derived from the adaptive immune system, using the bacterial CRISPR (clusters of regularly interspaced palindromic repeats)/Cas (CRISPR-associated) systems, has dramatically advanced in recent years. In this method, the Cas9 endonuclease, derived from Streptococcus pyogenes, and an artificially designed single-chain guide RNA (sgRNA) are used. The Cas9-sgRNA complex recognizes the protospacer-adjacent motif (5’-NGG-3’) and cleaves 3 bp upstream of the target site (Jinek et al., 2012). Subsequently, random insertion and/or deletion mutations occur during the repair process for double-strand breaks (DSBs) in the DNA. As targeted mutagenesis using these RGENs is efficient as well as cost- and time-effective, it has been used for genome editing in various organisms, including many plant species. Here, we established a protocol for targeted mutagenesis using RGENs in mosses, and demonstrated it in a model and a non-model species (Nomura et al., 2016).
Materials and Reagents
Equipment
Procedure
Data analysis
According to our evaluation based on the phenotypic change caused by the mutation of a target gene, the target mutagenesis efficiency in P. patens was approximately 45% to 68% (Nomura et al., 2016). Since efficiency is dependent on the target sequence, we recommend using the ‘focas’ website (http://focas.ayanel.com/; Doench et al., 2014; Xiao et al., 2014; Osakabe et al., 2016) for designing and predicting the on- and off-target efficiency of the target genome. In this case, the target sequence with an on-target score of 0.6 or higher and a low possibility of off-target effect is desirable.
Notes
Recipes
Acknowledgments
This protocol was adapted from a published paper (Nomura et al., 2016). This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B) (grant number 15K18824), and Grants-in-Aid for Scientific Research (C) (grant number 15K06905).
References
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