Crisper - A Gene editing tech.

Editor's Notes

• #6: Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis.
• #8: This figure shows three methods of plant genetic improvement. Traditional breeding crosses two parental lines that contain favorable traits, and plant scientists hope to find all of the desirable traits (and no undesirable traits) in one of the offspring. Many genes are mixed in random ways. Genetic engineering (what is commonly called “GMO”) adds one entire gene to an already elite genetic background. Gene editing only brings slight changes to the genetic code that is already there. The most minor adjustments are made with gene editing.
• #13: ZFNs are chimeric proteins containing multiple (usually 3–4) zinc finger DNA-binding domains and a DNA-cleavage domain (Fig. 2B). Each domain, comprising a stretch ofapproximately 30 amino acids stabilized by a zinc ion, can bind to a specific nucleotide triplet within the targeted DNA sequence (Durai et al., 2005). A DNA-cleavage domain, consisting of a FokI restriction enzyme site, is attached to the C-terminal end of this chimeric protein, comprising the first half of the ZFN pair, which is designed to recognize and bind to one strand of the target DNA sequence. The second half of the ZFN pair, whose structure is similar to that of the first half, is designed to recognize and bind to the opposite strand of the target DNA at a distance of six nucleotides from the first half of the ZFN pair. This six-nucleotide region acts as a spacer, which facilitates the dimerization of two inactive FokI DNA-cleavage domains to activate a functional nuclease used to create DSBs within this spacer region. Finally, the DSBs are repaired by an error-prone NHEJ mechanism, generating InDel mutations at the DSB site. ZFNs have been successfully used for gene editing in animals (Doyon et al., 2008; Perez et al., 2008) and plants (Lloyd
• #14: TALEN is an effector protein of TALEs (transcription activator-like effectors) secreted by the plant pathogenic bacteria Xanthomonas. The Xanthomonas inject TALE proteins into the host plant cell, where they enter the nucleus, bind to effector-specific DNA sequences in host gene promoters, and trigger transcription. This attribute of TALENs makes them efficient ENs for targeted gene editing. TALENs have emerged as an attractive alternative to ZFNs for use in TGE. Like ZFNs, TALENs consist of an N-terminal DNA-binding domain and a C-terminal nonspecific FokI nuclease domain. Their DNA-binding domain is composed of 13–28 monomers, each 34 amino acid residues in length. These 34 residues are highly conserved in each monomer, except the residues at positions 12 and 13, which are known as repeat variable di-residues (RVDs). The mostly common RVDs are HD, NG, NI, and NN, which play a major role in determining the binding affinities of TALENs by recognizing the C, T, A, and G nucleotides, respectively, in the target DNA of the TALENs. Similar to ZFNs, a pair of TALENs is required to create DSBs. The first-half of the TALEN pair is designed to recognize and bind to one strand, whereas the second half recognizes and binds to the opposite strand at a site 12–21 nucleotides away from the first binding site. Within this nucleotide spacer region, DSBs are produced upon dimerization of the two inactive FokI nuclease domains of each TALEN. Compared to ZFNs, TALENs have better binding affinity to their target sites through RVDs, making them efficient ENs for precise genome modification. Target gene modification via TALENs has been reported in plants including cereal crops (rice and wheat) and horticultural crops (tomato and potato) as well as animals, including zebrafish and human