**5.2 Gene editing**

The ease and efficiency of creating gene KO has improved recently through the use of engineered molecules to create genome disruptions in a process referred to as "gene editing". These novel molecules can be designed to generate double-strand DNA breaks at virtually any chosen genomic site *in situ*. Cellular machinery closely surveils the genome for double-strand breaks which are then recognized and often repaired by non-homologous end joining (NHEJ). Because NHEJ relies upon small single-strand overlaps at the ends of a break, the repair may be imprecise and, if within a coding region, can lead to frame shift mutations which inactivate the gene [44].

The most prominent of these novel tools for gene editing are Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), each of which consists of two regions: a sequence-specific DNA binding domain and an enzymatic function that creates a double-strand break in the target DNA [45–47]. For ZFN and TALEN, the synthetic DNA binding domain is created by repetitive protein modules which can be joined combinatorially to recognize a particular DNA sequence. Both approaches, while successful, require a significant investment of time and resources to identify functional molecules. CRISPR, like ZFN and TALEN, has the ability to generate double-strand DNA breaks, however, the DNA binding domain relies upon RNA base-pairing with target DNA for its precision. The use of an RNA to guide specificity greatly improves the speed and efficiency in identifying optimal molecules at a much lower cost, which has led to its rapid adoption in genome engineering [48, 49].

The use of CRISPR for the rapid modification of the pig genome was recently demonstrated with the ablation of porcine endogenous retroviral (PERV) sequences. The pig genome carries 25 or more copies of these gamma retroviral sequences, which are transmitted from parent to offspring through inheritance. Application of CRISPR was able to eliminate the PERV sequences from the genome of porcine cells [50]. Although the potential risk of infectious disease from porcine organs caused by PERV sequences in xenotransplantation is debatable, the results show the ability of CRISPR to target multiple, homologous loci throughout the genome. A key question that has arisen regarding large scale CRISPR targeting at multiple genomic sites is whether significant numbers of off-target double-stranded breaks were introduced, which may create unexpected mutations in the resulting pigs [51]. Nonetheless, the ease of use of CRISPR has resulted in widespread adoption for genome engineering in xenotransplantation.

Gene deletion has been instrumental in the advancement of xenotransplantation, however, there are limitations to its application; the genes of interest must be non-essential to pig viability, development, fertility and, most importantly, organ function. The number of distinct loci to be targeted is also a serious consideration, since independently-assorting alleles will be challenging to breed together in a reasonable timeframe. To address these concerns, additional engineering strategies are required as discussed below.
