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The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO·3KO·9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles.In vitroassays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO·3KO·9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism.One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

Homology-directed repair (HDR) maintains genomic integrity by eliminating lesions such as DNA double-strand breaks (DSBs), interstrand crosslinks (ICLs) and stalled replication forks and thus a deficiency in HDR is associated with genomic instability and cancer predisposition. The mechanism of HDR is best understood and most rigorously characterized in yeast. The inactivation of the fungal radiation sensitive 52 (RAD52) gene, which has both recombination mediator and single-strand annealing (SSA) activities in vitro, leads to severe HDR defects in vivo. Confusingly, however, the inactivation of murine and chicken RAD52 genes resulted in mouse and chicken cells, respectively, that were largely aphenotypic. To clarify this issue, we have generated RAD52 knockout human cell lines. Human RAD52-null cells retain a significant level of SSA activity demonstrating perforce that additional SSA-like activities must exist in human cells. Moreover, we confirmed that the SSA activity associated with RAD52 is involved in, but not absolutely required for, most HDR subpathways. Specifically, a deficiency in RAD52 impaired the repair of DNA DSBs and intriguingly decreased the random integration of recombinant adeno-associated virus (rAAV). Finally, an analysis of pan-cancer genome data from The Cancer Genome Atlas (TCGA) revealed an association between aberrant levels of RAD52 expression and poor overall survival in multiple cancers. In toto, our work demonstrates that RAD52 contributes to the maintenance of genome stability and tumor suppression in human cells.

Homology-directed genome editing is the intentional alteration of an endogenous genetic locus using information from an exogenous homology donor. A conversion tract is defined as the amount of genetic information that is converted from the homology donor to a given strand of the targeted chromosomal locus. Because of this, conversion tract analysis retrospectively not only elucidates the mechanism of homology-directed genome editing but also provides valuable insights on the conversion efficiency of every nucleotide in the homology donor. Here we describe a blue fluorescent protein-to-green fluorescent protein conversion system that can be conveniently used to measure the efficiency and analyze the lengths of conversion tracts of homology-directed genome editing using oligonucleotide donors in mammalian cells.

Homology-directed genome editing is the intentional alteration of an endogenous genetic locus using information from an exogenous homology donor. A conversion tract is defined as the amount of genetic information that is converted from the homology donor to a given strand of the targeted chromosomal locus. Because of this, conversion tract analysis retrospectively not only elucidates the mechanism of homology-directed genome editing but also provides valuable insights on the conversion efficiency of every nucleotide in the homology donor. Here we describe a blue fluorescent protein-to-green fluorescent protein conversion system that can be conveniently used to measure the efficiency and analyze the lengths of conversion tracts of homology-directed genome editing using oligonucleotide donors in mammalian cells.

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications., Precision genome engineering using homology donors and the endogenous DNA break repair machinery and recently CRISPR-Cas9 targeted APOBECs have been demonstrated. Here the authors design zinc-finger and TALE chimeric deaminases and investigate editing efficiency and off-target effects.

Nature Communications 7: Article number: 13330 (2016); Published 2 November 2016; Updated 9 October 2017. The original version of this Article contained an error in the email address of the corresponding author George Church. The correct email is gchurch@genetics.med.harvard.edu. The error has been corrected in the HTML and PDF versions of the Article.

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2—but not FANCI—plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. We previously demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs infect human cells, and we observe the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the value of PERV inactivation to prevent cross-species viral transmission and demonstrates the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation. This study is mainly funded by eGenesis Inc. and was funded by NIH grant P50 HG005550. Y.L. was funded by Danish Research Council for Independent Research (DFF-1337-00128) and Sapere Aude Young Research Talent Prize (DFF-1335-00763). M.G. was funded by a Human Frontiers Science Program Long Term fellowship. Some of the pig production was funded by Major Program on Basic Research Projects of Yunnan Province, China (Grant No. 2014FC006). PERV elements genotyping illumina miseq data have been uploaded to the European Nucleotide Archive (ENA) hosted by the European Bioinformatics Institute (EBI) with the submission reference PRJEB11222

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor.

Keeping sterile stocks or cultures of microalgae is fundamental to microalgae biotechnology as well as basic scientific research. However, contamination by bacteria and/or fungi in microalgae cultures or stocks is often a problem. Here, we have developed a strategy for reducing or eliminating bacterial and fungal contamination by using a cocktail of antibiotics. Chlamydomonas reinhardtii P. A. Dang., a widely used unicellular green alga, has been used as a testing organism. A combination of ampicillin, cefotaxime, and carbendazim removed or reduced contamination by three different bacteria and two different fungi tested. A step-by-step procedure is provided, which is simple, economical, and effective.

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection., Author Summary The repair of DNA damage is essential to maintain genomic stability. Cells have at least four distinct DNA repair pathways, and defects in any of them can lead to tumor formation and cancer progression. Herpes Simplex Virus-1 (HSV-1) manipulates components of the host DNA repair pathways. In this paper we showed that DNA repair by the single strand annealing (SSA) pathway was increased during HSV infection and that other pathways were inhibited. We also show that a viral nuclease in conjunction with either a viral or cellular single strand annealing protein can stimulate the SSA pathway. We suggest that viral DNA synthesis occurs via an SSAdependent mechanism that is reminiscent of that used by bacterial viruses such as λ. Interestingly, λ has evolved an SSA-mediated repair mechanism to exchange genetic information that has also been used to enhance gene targeting in bacteria. It is thus possible that HSV proteins could be similarly used as tools to stimulate gene targeting in human cells leading to more effective strategies for gene therapy. Furthermore, the diversity of HSV reported in human populations, combined with the high rate of genetic exchange during infection, suggests that SSA may play a role in viral evolution and pathogenesis.

Summary Flagella and cilia are structurally polarized organelles whose lengths are precisely defined, and alterations in length are related to several human disorders [1, 2]. Intraflagellar transport (IFT) and protein signaling molecules are implicated in specifying flagellar and ciliary length [3–6], but evidence has been lacking for a flagellum and cilium length sensor that could participate in active length control or establishment of structural polarity. Previously, we showed that the phosphorylation state of the aurora-like protein kinase CALK in Chlamydomonas is a marker of the absence of flagella. Here we show that CALK phosphorylation state is also a marker for flagellar length. CALK is phosphorylated in cells without flagella, and during flagellar assembly it becomes dephosphorylated. Dephosphorylation is not simply a consequence of initiation of flagellar assembly or of time after experimentally induced flagellar loss, but instead requires flagella to be assembled to a threshold length. Analysis of cells with flagella of varying lengths shows that the threshold length for CALK dephosphorylation is ∼6 μm (half length). Studies with short and long flagellar mutants indicate that cells detect absolute rather than relative flagellar length. Our results demonstrate that cells possess a mechanism for translating flagellar length into a posttranslational modification of a known flagellar regulatory protein.