Most of the xenotransplantation-related requests involve KO of GGTA1 (alpha 1,3 galactosyltransferase) and B4GALNT2 (beta-1,4-N-acetyl-galactosaminyl transferase 2). To produce the appropriate phenotype, an additional gene B4GALNT2L (LOC110255214: beta-1,4 N-acetylgalactosaminyltransferase 2-like) must also be disrupted. B4GALNT2 and B4GALNT2L are adjacent to one another on chromosome 12 and are separated by four unrelated genes that must be retained. The use of CRISPRs (or other editing strategies) must be used very carefully as to not remove the intervening 4 genes that lie between B4GALNT2 and B4GALTN2L. However, these genes are so intimately linked that heterozygotes are only useful if the KO alleles are on the same chromosome.
Most groups would also like to have a fourth gene knocked out eventually, CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase). However, for some non-human primate studies, they would prefer to retain a functional gene for now. Therefore, we have concluded that CMAH KO is of lower priority as compared to GGTA1, B4GALNT2 and B4GALTN2L.
Many groups also want the addition of a hCD46 transgene with ubiquitous expression. Although we have an hDAF transgene that works very well when placed at the GGTA1 locus, we have not yet assembled a CD46 counterpart. Since most groups favor CD46 over DAF, we are considering two strategies. In one strategy we add CD46 to our DAF expression cassette. In the other strategy we build a CD46 only transgene and place it at the GGTA1 locus. We welcome any input that your group may have related to these (or other) strategies.
We have Spy Cas9 guide RNAs that will routinely provide for disruption of all four of the genes above. However, we have not been able to produce fibroblast colonies that harbor all four modifications. Therefore, we are now shifting our efforts to produce the requested genotypes by sequential modification.
In parallel, we will pursue two strategies. Firstly, we will perform zygote injections with the reagents for B4GALNT2 and B4GALNT2L knockout as a primary effort. When these animals are born, they will be made available to the research community but will also be used for breeding purposes to backcross these knockouts onto the GGTA1 knockout genetics (+/- hDAF). Separately, a secondary effort will be used for CMAH when opportunities arise.
The second strategy will make use of fetal fibroblasts and “recloning” to sequentially add each modification until the desired genotype is obtained. If this strategy is successful, it will provide animals of the appropriate genotype in the shortest amount of time. However, propagation of the line will be problematic without inbreeding. In addition, we are likely to experience reduced efficiency at each round of recloning. Lastly, all animals produced in the first round will be cloned. The use of clones for experimental data is not recommended.