A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kilobases. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

Revising the Chromosome-Specific Probes of White Hawk (Leucopternis albicollis)

Leucopternis albicollis is a diurnal bird of prey with extensive karyotype reorganization. Chromosome-specific probes from this species have been used successfully to detect intrachromosomal rearrangements in different species of bird since 2010. However, some gaps were detected in this first set of probes. Here, we have obtained a new set of whole chromosome probes in order to improve the previous one; also, we have performed experiments using bacterial artificial chromosome (BAC) from chicken microchromosomes. Our results demonstrated that the microchromosomes were involved in fusion events. In addition, a new nomenclature has been proposed for the new set of probes and some inaccurate data were corrected.

Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

ABSTRACT Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo. This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

One of the Triple Poly(A) Signals in the M112-113 Gene Is Important and Sufficient for Stabilizing the M112-113 mRNA and the Replication of Murine Cytomegalovirus

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5′ terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3′ terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.

Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

Background Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone.Results Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days.Conclusion Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.