CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana
CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short
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Introduction Genome sequencing technologies have revolutionized the biological sciences. With the availability of genome sequences of various living organisms including plants, the focus is now shifted to uncover gene function [1]. In this regard, both forward and reverse genetic approaches have contributed immensely to plant functional genomics [2]. Geneticists initially used natural mutants to elucidate the function of genes, but later on artificial mutants were created by using physical, chemical, and biological agents. However, these methods have some limitations. For instance,
Daisuke Miki and Gaurav Zinta contributed equally with all other contributors. Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200, https://doi.org/10.1007/978-1-0716-0880-7_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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creating mutants by physical (radiation) and chemical (EMS, ethyl methanesulfonate) agents causes random mutations in the genome [3]. Thus, to establish a causal relationship between genotype and phenotype, large-scale genetic screening is required, which is labor intensive, costly, and time-consuming. The reverse genetics methods, including T-DNA insertion lines (e.g., SALK T-DNA insertion library), RNA interference (RNAi), and virus-induced gene silencing (VIGS), offer direct ways to elucidate gene function, which involve reduction of transcript levels of endogenous genes to generate knockdown mutants [4]. However, sometimes appropriate T-DNA insertion lines are not available in the libraries, the RNA silencing methods are not highly specific, and reductions achieved in the gene expression are variable and not stably inherited to next generation. Sequence-specific nucleases (SSNs) generate target site-specific double strand breaks (DSBs) in the genome of numerous organisms [5–8]. Due to this property, SSNs including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) have emerged as versatile tools for genome engineering. Particularly, CRISPR/Cas9 system is the most frequently used genome editing tool in plants due to its simplicity, high specificity, efficiency, and multiplexing capacity [9]. The Cas9 endonuclease is directed to specific genomic loci by a 20-nt single-guide RNA (sgRNA) (Fig. 1a). Type II Streptococcus pyogenes Cas9 (SpCas9) is the most widely used Cas9 that recognize NGG as a protospacer adjacent motif (PAM) sequence. The activity of Cas9 generates a double strand break (DSB) at 3–4 base pairs distal to the PAM sequence (Fig. 1b). DSBs are subsequently repaired by either error-prone nonhomologous end joining (NHEJ) or error-free homologydirected repair (HDR), if certain homologous DNA repair donor template is provided, resulting in gene mutations or knock-in/ replacement, respectively (Fig. 1a). The imprecise repair of DSBs by NHEJ causes ra
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