However, traditional cloning, based on restriction-ligation reactions or two-round overlapping PCRs, is time-consuming. The sgRNA expression cassette can then be introduced into a binary vector via Golden Gate cloning or Gibson Assembly (Ma et al., 2016). In an alternative approach, an overlapping PCR, with two rounds of reactions, can also establish a sgRNA expression cassette with adaptors.
![snapgene overlapping primers snapgene overlapping primers](https://www.snapgene.com/wordpress/wp-content/uploads/2018/12/Uniqueness-600x337.png)
In this approach, a restriction-ligation reaction inserts a spacer into intermediate vectors to produce an sgRNA expression cassette, which fuses with adaptors for Golden Gate cloning or Gibson Assembly (Ma et al., 2016). (2016) have developed this basic system into a CRISPR/Cas9 system for multiplex genome editing in rice. Then, an “LR Clonase” reaction transfers the target-sgRNA cassette into a destination clone, which contains a Cas9 expression cassette (Feng et al., 2013). In a restriction-ligation reaction, a gene-specific sgRNA spacer substitutes the target region in the entry clone, which encodes attL recombination sites. (2013) have constructed gateway vectors to co-express Cas9 and sgRNAs in plants through Agrobacterium sp.-mediated transformation. CRISPR – binary vectors express two elements – the sgRNAs with a target sequence (target-sgRNAs) and Cas9 protein – to cleave target genomic regions. Researchers have now developed this system into a key tool for targeted genome editing. Keywords: Cloning system, Multiplex PCR, CRISPR/Cas9, Genome editing, Riceīacteria defend against viruses through a protein system, consisting of the clustered regularly interspaced short palindromic repeat (CRISPR), the CRISPR-associated (Cas) protein, CRISPR RNAs (crRNAs) and trans-encoded crRNA (tracrRNA). In this protocol, we describe the detailed step-by-step instructions for using this system. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. We also amplified two sgRNA expression cassettes through a single round of PCR. In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020).
![snapgene overlapping primers snapgene overlapping primers](https://wiki.bits.vib.be/images/2/2b/SG60.png)
However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. CRISPR/Cas9 is an established and flexible tool for genome editing.