Abstract
CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.
Highlights
Since gene-knockout technology has become available in plant research, reverse genetics is widely used to establish the logic for proving the expected hypothesis
The protocol in this study describes the construction of a multi-single guide RNA (sgRNA) vector that contains at least four sgRNAs, using the common clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 vector based on type IIS restriction enzyme ligation
Fatty acid desaturase 2 (FAD2) and fatty acyl-acyl carrier protein (ACP) thioesterase B (FATB), which exist as multicopy genes in the soybean genome, need to be knocked out
Summary
Since gene-knockout technology has become available in plant research, reverse genetics is widely used to establish the logic for proving the expected hypothesis. Reverse genetics is used to elucidate hypotheses by knocking out or modifying a gene of interest (GOI) and examining the altered phenotype of a model species [1]. Advances in biotechnology have led to the development of many methods for manipulating GOIs, of which the most frequently used are zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) before the advent of clustered regulatory interspaced short palindromic repeats (CRISPR) systems [1]. All these programmable nucleases can cause double-strand breaks (DSB) and induce DNA repair systems such as homologous recombination (HR) or non-homologous end-joining (NHEJ), which fundamentally reform or delete original base sequences. The sgRNA is a 20-nucleotide long, single-stranded RNA that combines with the Cas protein and provides information about the target site through a complementary sequence
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