**Author details**

#### Sean Stevens

**10.3. Tools for genome engineering**

346 Organ Donation and Transplantation - Current Status and Future Challenges

has been greatly facilitated [70].

**11. Conclusions and future prospects**

A number of novel enzymatic molecules have been created which help resolve the dilemma of targeted integration in porcine cells. Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are all synthetic molecules based on genuine proteins which allow the precise targeting of genomic DNA based upon sequence [66–68]. In each case there are two functional components, a targeting module which recognizes a specific genomic sequence, and an enzymatic module which introduces a double-stranded DNA break at the target site. In the case of ZFN and TALEN, the targeting module is a complex array of protein sequences which have previously been shown to recognize specific DNA sequences and can be mixed in a modular way to bind to any desired sequence. Although both technologies have shown great success, the effort and cost required to identify a single functional molecule can be significant. In contrast, the relatively more recently recognized CRISPR, and related prokaryotic systems, is much more easily applied in mammalian cells. The DNA binding module in this case is RNA base-pairing to provide sequence specificity. The enzymatic module cleaves DNA, creating a double strand break similar to ZFN and TALEN. When heterologous DNA is present, the cellular repair machinery may use the synthetic DNA to repair the break, inserting the TG at the desired genomic site. It is important to note that all of these systems, ZFN, TALEN or CRISPR, are essentially the same in that they introduce double strand DNA breaks at a selected site in the genome and do not directly affect the rate of DNA insertion. Therefore, it is often necessary to include selection schemes for identification of the modified cells. The greater efficiency and ease of use of these systems, CRISPR in particular, has allowed targeted

insertion of DNA into genomes that were not previously able to be modified [69].

Due to the challenges of creating genomic modifications in porcine primary cells while maintaining their viability for SCNT, more efficient engineering methods are desirable. One approach to enhance efficiency is to target a specific region of DNA, called a landing pad, with multiple genes at once. By inserting a DNA vector bearing multiple therapeutic genes at once, a large amount of breeding and testing can be circumvented using a single event. This approach has the added advantage of avoiding inefficient crossbreeding necessary to bring loci from distinct chromosomes together in one lineage. When combined with the use of tools such as ZFN, TALEN and CRISPR more rapid progress in the genetic modification of animals

The increasing sophistication and accessibility of genome engineering toolsets and deeper understanding of immunological rejection mechanisms has allowed greater advancement in xenotransplantation than ever before. A key question is just how many genetic changes are required in order to make a pig organ suitable for transplantation? While the critical experimental data needed for such an assessment is still accumulating, it is clear that the number of alterations required for one organ may be different from another. For example, xeno-hearts with relatively minimal genetic modifications have demonstrated months to years survival in transplantation studies with non-human primates, whereas xeno-lungs with more extensive modifications have Address all correspondence to: sean\_m\_stevens@yahoo.com

Synthetic Genomics, Inc., La Jolla, CA, USA
