*1.1. The Discovery and Development of CRISPR Technology*

*CRISPR-associated* (Cas) genes were first discovered in the *Escherichia coli* genome in 1987 and were officially named by the Dutch scientist who identified them [12]. In 2005, it was discovered that many CRISPR spacers consist of short sequences that are highly homologous with sequences originating from extrachromosomal DNA. The *Cas*-encoded protein can combine with the CRISPR transcription products and with the homologous foreign DNA sequences to form a protein–RNA complex, which can cut the foreign DNA fragments. The primary function of the CRISPR complex in bacteria and archaea is to integrate specific fragments of exogenous DNA (from invading phages or other sources) into their own genomes to become interval sequences. During subsequent invasion by foreign DNA, the specific recognition system is then activated, providing an acquired immune defence function [13–15].

CRISPR-Cas technology has been successfully applied to the editing of human, animal, and plant genomes, and has been developed for use in drug screening, animal domestication, and food science research [16–18]. There are three main types of CRISPR-Cas systems. Types I and III use a large multi-Cas protein complex for interference [19]. Type II requires only a simple effector-module architecture to accomplish interference via its two signature nuclease domains, RuvC and HNH [20]. Among various CRISPR nucleases, type II Cas9 from *Streptococcus pyogenes* (SpCas9) is the most widely used in CRISPR-Cas technology [21]. The sgRNA-Cas complex recognises the protospacer adjacent motif (PAM) and Cas9 cleaves the target DNA to generate a double-strand break (DSB), triggering cellular DNA repair mechanisms (Figure 1). In eukaryotes, DSBs have two main repair mechanisms. The first is nonhomologous end joining (NHEJ). In the absence of a homologous repair template, the NHEJ repair pathway is activated at the DSB site, thus disrupting gene function. The second is homology directed repair (HDR). If a donor DNA template homologous to the sequence surrounding the DSB site is available, the HDR pathway is initiated, precisely introducing specific mutations such as insertion or replacement of desired sequences into the break sites [22]. Using a donor DNA as a template, gene targeting (GT) can precisely modify a target locus to repair DNA DSBs.

Several strategies are used to improve the homologous recombination frequency between a genomic target and an exogenous homologous template donor. Most of the strategies focus on enhancing the number of donor repair templates using virus replicons [23], suppressing the NHEJ pathway [24], and timing DSB induction at target sites to coincide with donor repair template delivery in plant cells [25]. Finally, the recombination frequency can be enhanced by treatment with Rad51-stimulatory compound1 (RS-1) [26].

**Figure 1.** The potential applications of CRISPR-Cas systems in genome editing. CRISPR-Cas systems mediated genome modification depending on the two main double-strand break (DSB) repair pathways. Indel mutation and gene deletion are outcomes of the dominant nonhomologous end joining (NHEJ) repair pathway. Gene insertion, correction, and replacement, using a DNA donor template, are outcomes of the homology directed repair (HDR) pathway.
