Mega-nucleases The first tools to be used for genome editing, mega-nucleases are naturally occurring enzymes found in bacteria. One single region on the mega-nuclease recognizes and binds to relatively long DNA sequences (14- 40 nucleotides long), then cleaves the DNA (Yee, 2016, Zhu et al., 2016). Since all of the activities are located within one protein domain, it is difficult to separate the targeting and DNA cutting functions of mega-nucleases and thus it is impossible to program the nuclease to target new sites on the genome for cleavage. Since the sequence recognition sites for mega-nucleases that have been identified so far do not occur naturally in the plant genome, there are limits to how useful they are for genome editing in crops.
4b. Zinc Finger Nucleases (ZFN) Zinc finger nucleases are hybrid proteins consisting of a DNA binding domain (consisting of three or four binding modules, with each module recognizing a specific segment of DNA) that has been fused to a nuclease domain, which creates a DNA break (Wang et al., 2016, Zhu, 2016). ZFNs can be cumbersome to design, and can have some off-target effects, meaning that they can bind to additional unintended sites and cleave DNA at locations other than the one desired. Another disadvantage of using ZFN is the high cost of licensing the technology.
4c. TALENs As a technology, TALENs utilize the transcriptional activator-like effector (TALE) protein derived from the bacteria Xanthomonas as its DNA binding domain. This TALE DNA binding domain is fused to a nuclease domain (Benjamin et al.,m 2016). Since the target recognition sequence is larger for TALENs than for ZFNs, TALEN-based technologies display fewer off-target effects, meaning that the DNA binding domain binds exactly to the target site and nowhere else on the genome. A drawback to the use of TALENsS is the difficulty of assembling the DNA binding domain (Merkert and Martin, 2016).
4d. CRISPR/Cas9. CRISPR-Cas9 has rapidly become the main tool for genome editing in plant science research laboratories. Discovered first in a common bacterium found in the intestinal tract, CRISPR-Cas9 is composed of a ribonucleoprotein complex containing both a CRISPR (clustered regularly interspaced short palindromic repeat) sequence of RNA) and a Cas (CRISPR-associated) protein that protects bacteria from invading bacteriophage DNA (Bono et al., 2015, Quetre, 2016).
For a long time, short DNA repeats that are interspaced with sequences containing homology to virus sequences (known as CRISPR loci) have been observed in the genomes of bacteria. Adjacent to these virus sequences are genes encoding a series of Cas proteins (Wang and Qi, 2016). CRISPR loci and Cas proteins play a unique role in the bacteria’s defence mechanism against invading pathogens; the bacteria can recognize a particular virus that infects the cell based on homology with one of its CRISPR loci. The relevant sequence can then be used as guide RNA to direct the Cas system to destroy the invading virus by destroying its genetic material. Cas9 is a protein within the cas repertoire which can actually cleave DNA at the target site proposed by the CRISPR loci.
Researchers soon discovered that Cas9 could be easily adapted for use in genome editing and began to make their own versions of CRISPR synthetic guide RNA (sgRNA) that could be targeted to any sequence of any organism. The CRISPR RNA molecule is able to guide the nuclease to a specific DNA target site, at which the Cas9 nuclease performs its cleavage function (Sander and Joung, 2014). Since Cas9 is efficient at causing a highly specific cleavage event within a target sequence of about 20 nucleotides, it is much easier to create sgRNAs than it is to form specific binding domains on proteins that ZFN or TALEN-based technologies require. The cell’s repair machinery then makes the desired permanent change in the genome. The technology is versatile, available, and easy to use. While some off target cleavage was originally reported upon the first applications of CRISPR- Cas9, this has been substantially reduced by altering the Cas9: sgRNA ratio and also by using computer software that assists in sgRNA design and reduces the potential for off target effects.
In addition to its use as a genome editing tool, the targeting function of CRISPR-Cas9 has made it an effective tool at localizing gene expression. This can be achieved by linking an inactivated version of Cas9 to a fluorescent protein. Furthermore, Cas9 can be fused to proteins that activate or suppress a variety of genes, and targeted to any regulatory element on a genome.