Faculty Bibliography

Due to the sheer number of COVID-19 (coronavirus disease 2019) cases, the prevalence of asymptomatic cases and the fact that undocumented cases appear to be significant for transmission of the causal virus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), there is an urgent need for increased SARS-CoV-2 testing capability that is both efficient and effective ¹ . In response to the growing threat of the COVID-19 pandemic in February, 2020, the FDA (US Food and Drug Administration) began issuing Emergency Use Authorizations (EUAs) to laboratories and commercial manufacturers for the development and implementation of diagnostic tests ¹ . So far, the gold standard assay for SARS-CoV-2 detection is the RT-qPCR (real-time quantitative polymerase chain reaction) test ² . However, the authorized RT-qPCR test protocols vary widely, not only in the reagents, controls, and instruments they use, but also in the SARS-CoV-2 genes they target, what results constitute a positive SARS-CoV-2 infection, and their limit of detection (LoD). The FDA has provided a web site that lists most of the tests that have been issued EUAs, along with links to the authorization letters and summary documents describing these tests ¹ . However, it is very challenging to use this site to compare or replicate these tests for a variety of reasons. First, at least 12 of 18 tests that were issued EUAs prior to March 31, 2020, are not listed there. Second, the data are not standardized and are only provided as longhand prose in the summary documents. Third, some details (e.g. primer sequences) are absent from several of the test descriptions. Fourth, for tests provided by commercial manufacturers, summary documents are completely missing. To address at least the first three issues, we have developed a database, EUAdb (EUAdb.org), that holds standardized information about EUA-issued tests and is focused on RT-qPCR diagnostic tests, or "high complexity molecular-based laboratory developed tests" ¹ . By providing a standardized ontology and curated data in a relational architecture, we seek to facilitate comparability and reproducibility, with the ultimate goal of consistent, universal and high-quality testing nationwide. Here, we document the basics of the EUAdb data architecture and simple data queries. The source files can be provided to anyone who wants to modify the database for his/her own research purposes. We ask that the original source of the files be made clear and that the database not be used in its original or modified forms for commercial purposes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.

Long interspersed element-1 (LINE-1, L1) sequences, which comprise about 17% of human genome, are the product of one of the most active types of mobile DNAs in modern humans. LINE-1 insertion alleles can cause inherited and de novo genetic diseases, and LINE-1-encoded proteins are highly expressed in some cancers. Genome-wide LINE-1 mapping in single cells could be useful for defining somatic and germline retrotransposition rates, and for enabling studies to characterize tumour heterogeneity, relate insertions to transcriptional and epigenetic effects at the cellular level, or describe cellular phylogenies in development. Our laboratories have reported a genome-wide LINE-1 insertion site mapping method for bulk DNA, named transposon insertion profiling by sequencing (TIPseq). There have been significant barriers applying LINE-1 mapping to single cells, owing to the chimeric artefacts and features of repetitive sequences. Here, we optimize a modified TIPseq protocol and show its utility for LINE-1 mapping in single lymphoblastoid cells. Results from single-cell TIPseq experiments compare well to known LINE-1 insertions found by whole-genome sequencing and TIPseq on bulk DNA. Among the several approaches we tested, whole-genome amplification by multiple displacement amplification followed by restriction enzyme digestion, vectorette ligation and LINE-1-targeted PCR had the best assay performance. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.

LINE-1 retrotransposon overexpression is a hallmark of human cancers. We identified a colorectal cancer wherein a fast-growing tumor subclone downregulated LINE-1, prompting us to examine how LINE-1 expression affects cell growth. We find that nontransformed cells undergo a TP53-dependent growth arrest and activate interferon signaling in response to LINE-1. TP53 inhibition allows LINE-1⁺ cells to grow, and genome-wide-knockout screens show that these cells require replication-coupled DNA-repair pathways, replication-stress signaling and replication-fork restart factors. Our findings demonstrate that LINE-1 expression creates specific molecular vulnerabilities and reveal a retrotransposition-replication conflict that may be an important determinant of cancer growth.

The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1- encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy and paste" mechanism. ORF1p is an RNA-binding protein and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p, only 16% of trialanine mutants retained near-wild-type activity. All ORF1p mutants formed near-wild-type levels of mRNA transcripts and seventy-five percent formed near-wild-type levels of protein. Two ORF1p mutants present a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis, but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins and target DNA throughout the L1 life cycle.