SUMMARY The “CTCF code” hypothesis posits that CTCF pleiotropic functions are driven by recognition of diverse sequences through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is supported by in vitro binding studies of a limited number of sequences. To study CTCF multivalency in vivo, we define ZF binding requirements at ~50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs 4–7 anchor CTCF to ~80% of targets containing the core motif. Nonconserved flanking sequences are recognized by ZFs 1–2 and ZFs 8–11 clusters, which also stabilize CTCF broadly. Alternatively, ZFs 9–11 associate with a second phylogenetically conserved upstream motif at ~15% of its sites. Individually, ZFs increase overall binding and chromatin residence time. Unexpectedly, we also uncovered a conserved downstream DNA motif that destabilizes CTCF occupancy. Thus, CTCF associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-Seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly-transcribed genes, including Myc and Pim1 cell cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

SUMMARY The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

SUMMARY The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of “cryptic” recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

Background Large skull reconstruction, with the use of customized cranial implants, restores cerebral protection, physiologic homeostasis, and one's preoperative appearance. Cranial implants may be composed of either bone or a myriad of alloplastic biomaterials. Recently, patient-specific cranial implants have been fabricated using clear polymethylmethacrylate (PMMA), a visually transparent and sonolucent variant of standard opaque PMMA. Given the new enhanced diagnostic and therapeutic applications of clear PMMA, we present here a study evaluating all outcomes and complications in a consecutive patient series. Methods A single-surgeon, retrospective, 3-year study was conducted on all consecutive patients undergoing large cranioplasty with clear PMMA implants (2016–2019). Patients who received clear PMMA implants with embedded neurotechnologies were excluded due to confounding variables. All outcomes were analyzed in detail and compared with previous studies utilizing similar alloplastic implant materials. Results Fifty-five patients underwent cranioplasty with customized clear PMMA implants. Twenty-one (38%) were performed using a single-stage cranioplasty method (ie, craniectomy and cranioplasty performed during the same operation utilizing a prefabricated, oversized design and labor-intense, manual modification), whereas the remaining 34 (62%) underwent a standard, 2-stage reconstruction (craniectomy with a delayed surgery for cranioplasty and minimal-to-no implant modification necessary). The mean cranial defect size was 101.8 cm2. The mean follow-up time was 9 months (range, 1.5–39). Major complications requiring additional surgery occurred in 7 patients (13%) consisting of 2 (4%) cerebrospinal fluid leaks, 2 (4%) epidural hematomas, and 3 (4%) infections. In addition, 3 patients developed self-limiting or nonoperative complications including 2 (4%) with new onset seizures and 1 (2%) with delayed scalp healing. Conclusions This is the first reported consecutive case series of cranioplasty reconstruction using customized clear PMMA implants, demonstrating excellent results with regard to ease of use, safety, and complication rates well below published rates when compared with other alloplastic materials. Clear PMMA also provides additional benefits, such as visual transparency and sonolucency, which is material specific and unavailable with autologous bone. Although these early results are promising, further studies with multicenter investigations are well justified to evaluate long-term outcomes.

The COVID-19 pandemic has resulted in an unparalleled need for viral testing capacity across the world and is a critical requirement for successful re-opening of economies. The logistical barriers to near-universal testing are considerable. We have designed an injection molded polypropylene anterior nares swab, the Rhinostic, with a screw cap integrated into the swab handle that is compatible with fully automated sample accessioning and processing. The ability to collect and release both human and viral material is comparable to that of several commonly used swabs on the market. SARS-CoV-2 is stable on dry Rhinostic swabs for at least 3 days, even at 42 °C, and elution can be achieved with small volumes. To test the performance of the Rhinostic in patients, 119 samples were collected with Rhinostic and the positive and negative determinations were 100% concordant with samples collected using Clinical Laboratory Improvement Amendments (CLIA) use approved nasal swabs at a clinical lab. The Rhinostic swab and barcoded tube set can be produced, sterilized, and packaged cost effectively and is designed to be adopted by clinical laboratories using automation to increase throughput and dramatically reduce the cost of a standard SARS-CoV-2 detection pipeline.