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A 57-year-old man with nonischemic cardiomyopathy who was dependent on venoarterial extracorporeal membrane oxygenation (ECMO) and was not a candidate for standard therapeutics, including a traditional allograft, received a heart from a genetically modified pig source animal that had 10 individual gene edits. Immunosuppression was based on CD40 blockade. The patient was weaned from ECMO, and the xenograft functioned normally without apparent rejection. Sudden diastolic thickening and failure of the xenograft occurred on day 49 after transplantation, and life support was withdrawn on day 60. On autopsy, the xenograft was found to be edematous, having nearly doubled in weight. Histologic examination revealed scattered myocyte necrosis, interstitial edema, and red-cell extravasation, without evidence of microvascular thrombosis — findings that were not consistent with typical rejection. Studies are under way to identify the mechanisms responsible for these changes. (Funded by the University of Maryland Medical Center and School of Medicine.) In this report, a porcine-to-human heart transplantation is described. Videos show transthoracic echocardiography in the patient who received the heart.

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae. The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro,with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.

The increasing burden of dengue, and the relative failure of traditional vector control programs highlight the need to develop new control methods. SIT using self-limiting genetic technology is one such promising method. A self-limiting strain of Aedes aegypti, OX513A, has already reached the stage of field evaluation. Sustained releases of OX513A Ae. aegypti males led to 80% suppression of a target wild Ae. aegypti population in the Cayman Islands in 2010. Here we describe sustained series of field releases of OX513A Ae. aegypti males in a suburb of Juazeiro, Bahia, Brazil. This study spanned over a year and reduced the local Ae. aegypti population by 95% (95% CI: 92.2%-97.5%) based on adult trap data and 81% (95% CI: 74.9-85.2%) based on ovitrap indices compared to the adjacent no-release control area. The mating competitiveness of the released males (0.031; 95% CI: 0.025-0.036) was similar to that estimated in the Cayman trials (0.059; 95% CI: 0.011-0.210), indicating that environmental and target-strain differences had little impact on the mating success of the OX513A males. We conclude that sustained release of OX513A males may be an effective and widely useful method for suppression of the key dengue vector Ae. aegypti. The observed level of suppression would likely be sufficient to prevent dengue epidemics in the locality tested and other areas with similar or lower transmission.

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

Expressing guide RNAs from a tRNA scaffold enhances mutagenesis by both Cas9 and Cpf1 and allows conditional CRISPR applications in vivo . We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila . The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA–sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes 1 . It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. Here we show that deep learning models 2 – 6 , can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. We evaluate the function of fully synthetic enhancers to specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. We further exploit enhancer design to create ‘dual-code’ enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, we characterize enhancer codes through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, we apply the same strategies to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states. Deep learning models were used to design synthetic cell-type-specific enhancers that work in fruit fly brains and human cell lines, an approach that also provides insights into these gene regulatory elements.

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.