Voices of the Costa Rican scientific diaspora: Policy lessons from a decade of experiences from our scientists abroad.

Scientific diasporas have been identified as valuable resources to strengthen science, technology, and innovation in their countries of origin. In this context, our paper seeks to contribute by addressing the following research questions: What are the main features of the Costa Rican scientific diaspora, and what policy lessons can be extracted from their experiences abroad? Toward this goal, we analyzed ten years of diaspora perspectives as collected by TicoTal, an online database and network of Costa Rican scientists studying and working abroad created by the National Academy of Sciences (ANC) in 2010. Our study reveals the main features of the Costa Rican scientific diaspora using 121 interviews published over a ten-year period: we identified the academic areas in which the diaspora has specialized, the countries where they were trained, their current location, the most frequent funding mechanisms and sources that enabled professional opportunities abroad, the level of engagement and collaboration they maintain with the Costa Rican STI ecosystem, along with the incentives they consider important to support and harness the potential of this community to advance STI goals in the country. Results from this analysis can inform national policies and investment strategies in R&D infrastructure and resources, by providing a roadmap to engage with scientific diasporas and benefit from their training and talent, as well as guide future scholarship and exchange programs.

Decorating chromatin for enhanced genome editing using CRISPR-Cas9.

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

p53 mediates target gene association with nuclear speckles for amplified RNA expression.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

DNA capture by a CRISPR-Cas9-guided adenine base editor.

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

Gcn5-Mediated Histone Acetylation Governs Nucleosome Dynamics in Spermiogenesis.

During mammalian spermatogenesis, germ cell chromatin undergoes dramatic histone acetylation-mediated reorganization, whereby 90%-99% of histones are evicted. Given the potential role of retained histones in fertility and embryonic development, the genomic location of retained nucleosomes is of great interest. However, the ultimate position and mechanisms underlying nucleosome eviction or retention are poorly understood, including several studies utilizing micrococcal-nuclease sequencing (MNase-seq) methodologies reporting remarkably dissimilar locations. We utilized assay for transposase accessible chromatin sequencing (ATAC-seq) in mouse sperm and found nucleosome enrichment at promoters but also retention at inter- and intragenic regions and repetitive elements. We further generated germ-cell-specific, conditional knockout mice for the key histone acetyltransferase Gcn5, which resulted in abnormal chromatin dynamics leading to increased sperm histone retention and severe reproductive phenotypes. Our findings demonstrate that Gcn5-mediated histone acetylation promotes chromatin accessibility and nucleosome eviction in spermiogenesis and that loss of histone acetylation leads to defects that disrupt male fertility and potentially early embryogenesis.

p63 establishes epithelial enhancers at critical craniofacial development genes.

The transcription factor p63 is a key mediator of epidermal development. Point mutations in p63 in patients lead to developmental defects, including orofacial clefting. To date, knowledge on how pivotal the role of p63 is in human craniofacial development is limited. Using an inducible transdifferentiation model, combined with epigenomic sequencing and multicohort meta-analysis of genome-wide association studies data, we show that p63 establishes enhancers at craniofacial development genes to modulate their transcription. Disease-specific substitution mutation in the DNA binding domain or sterile alpha motif protein interaction domain of p63, respectively, eliminates or reduces establishment of these enhancers. We show that enhancers established by p63 are highly enriched for single-nucleotide polymorphisms associated with nonsyndromic cleft lip ± cleft palate (CL/P). These orthogonal approaches indicate a strong molecular link between p63 enhancer function and CL/P, illuminating molecular mechanisms underlying this developmental defect and revealing vital regulatory elements and new candidate causative genes.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis.

Epithelial tissues rely on a highly coordinated balance between self-renewal, proliferation, and differentiation, disruption of which may drive carcinogenesis. The epigenetic regulator KMT2D (MLL4) is one of the most frequently mutated genes in all cancers, particularly epithelial cancers, yet its normal function in these tissues is unknown. Here, we identify a novel role for KMT2D in coordinating this fine balance, as depletion of KMT2D from undifferentiated epidermal keratinocytes results in reduced proliferation, premature spurious activation of terminal differentiation genes, and disorganized epidermal stratification. Genome-wide, KMT2D interacts with p63 and is enriched at its target enhancers. Depletion of KMT2D results in a broad loss of enhancer histone modifications H3 Lys 4 (H3K4) monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) as well as reduced expression of p63 target genes, including key genes involved in epithelial development and adhesion. Together, these results reveal a critical role for KMT2D in the control of epithelial enhancers and p63 target gene expression, including the requirement of KMT2D for the maintenance of epithelial progenitor gene expression and the coordination of proper terminal differentiation.

Def1 interacts with TFIIH and modulates RNA polymerase II transcription.

The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.

Comprehensive analysis of histone post-translational modifications in mouse and human male germ cells.

BACKGROUND: During the process of spermatogenesis, male germ cells undergo dramatic chromatin reorganization, whereby most histones are replaced by protamines, as part of the pathway to compact the genome into the small nuclear volume of the sperm head. Remarkably, approximately 90 % (human) to 95 % (mouse) of histones are evicted during the process. An intriguing hypothesis is that post-translational modifications (PTMs) decorating histones play a critical role in epigenetic regulation of spermatogenesis and embryonic development following fertilization. Although a number of specific histone PTMs have been individually studied during spermatogenesis and in mature mouse and human sperm, to date, there is a paucity of comprehensive identification of histone PTMs and their dynamics during this process. RESULTS: Here we report systematic investigation of sperm histone PTMs and their dynamics during spermatogenesis. We utilized "bottom-up" nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) to identify histone PTMs and to determine their relative abundance in distinct stages of mouse spermatogenesis (meiotic, round spermatids, elongating/condensing spermatids, and mature sperm) and in human sperm. We detected peptides and histone PTMs from all four canonical histones (H2A, H2B, H3, and H4), the linker histone H1, and multiple histone isoforms of H1, H2A, H2B, and H3 in cells from all stages of mouse spermatogenesis and in mouse sperm. We found strong conservation of histone PTMs for histone H3 and H4 between mouse and human sperm; however, little conservation was observed between H1, H2A, and H2B. Importantly, across eight individual normozoospermic human semen samples, little variation was observed in the relative abundance of nearly all histone PTMs. CONCLUSION: In summary, we report the first comprehensive and unbiased analysis of histone PTMs at multiple time points during mouse spermatogenesis and in mature mouse and human sperm. Furthermore, our results suggest a largely uniform histone PTM signature in sperm from individual humans.

New Synthetic Routes to Triazolo-benzodiazepine Analogues: Expanding the Scope of the Bump-and-Hole Approach for Selective Bromo and Extra-Terminal (BET) Bromodomain Inhibition.

We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra-terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl rings, in addition to the diazepine ternary center and the side chain methylene moiety. Substitution at the level of the side chain methylene afforded compounds targeting specifically and potently engineered BET bromodomains designed as part of a bump and hole approach. We further demonstrate that marked selectivity for the second over the first bromodomain can be achieved with an indole derivative that exploits differential interaction with an aspartate/histidine conservative substitution on the BC loop of BET bromodomains.

Chemical biology. A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes.

Small molecules are useful tools for probing the biological function and therapeutic potential of individual proteins, but achieving selectivity is challenging when the target protein shares structural domains with other proteins. The Bromo and Extra-Terminal (BET) proteins have attracted interest because of their roles in transcriptional regulation, epigenetics, and cancer. The BET bromodomains (protein interaction modules that bind acetyl-lysine) have been targeted by potent small-molecule inhibitors, but these inhibitors lack selectivity for individual family members. We developed an ethyl derivative of an existing small-molecule inhibitor, I-BET/JQ1, and showed that it binds leucine/alanine mutant bromodomains with nanomolar affinity and achieves up to 540-fold selectivity relative to wild-type bromodomains. Cell culture studies showed that blockade of the first bromodomain alone is sufficient to displace a specific BET protein, Brd4, from chromatin. Expansion of this approach could help identify the individual roles of single BET proteins in human physiology and disease.