Correction of the pathogenic mutation in TGM1 gene by adenine base editing in mutant embryos.

A couple diagnosed as carriers for lamellar ichthyosis, an autosomal recessive rare disease, encountered two pregnancy losses. Their blood samples showed the same heterozygous c.607C>T mutation in the TGM1 gene. However, we found that about 98.4% of the sperm had mutations, suggesting possible de novo germline mutation. To explore the probability of correcting this mutation, we used two different adenine base editors (ABEs) combined with related truncated single guide RNA (sgRNA) to repair the pathogenic mutation in mutant zygotes. Our results showed that the editing efficiency was 73.8% for ABEmax-NG combined with 20-bp-length sgRNA and 78.7% for Sc-ABEmax combined with 19-bp-length sgRNA. The whole-genome sequencing (WGS) and deep sequencing analysis demonstrated precise DNA editing. This study reveals the possibility of correcting the genetic mutation in embryos with the ABE system.

Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.

Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.

Putative roles as oncogene or tumour suppressor of the Mid-clustered microRNAs in Gallid alphaherpesvirus 2 (GaHV2) induced Marek's disease lymphomagenesis.

In the last decade, numerous microRNAs (miRNAs) have been identified in diverse virus families, particularly in herpesviruses. Gallid alphaherpesvirus 2 (GaHV2) is a representative oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts, namely Marek's disease (MD). In the GaHV2 genome there are 26 mature miRNAs derived from 14 precursors assembled into three clusters, namely the Meq-cluster, Mid-cluster and LAT-cluster. Several GaHV2 miRNAs, especially those in the Meq-cluster (e.g. miR-M4-5p), have been demonstrated to be critical in MD pathogenesis and/or tumorigenesis. Interestingly the downstream Mid-cluster is regulated and transcribed by the same promoter as the Meq-cluster in the latent phase of the infection, but the role of these Mid-clustered miRNAs in GaHV2 biology remains unclear. We have generated the deletion mutants of the Mid-cluster and of its associated individual miRNAs in GX0101 virus, a very virulent GaHV2 strain, and demonstrated that the Mid-clustered miRNAs are not essential for virus replication. Using GaHV2-infected chickens as an animal model, we found that, compared with parental GX0101 virus, the individual deletion of miR-M31 decreased the mortality and gross tumour incidence of infected chickens while the deletion individually of miR-M1 or miR-M11 unexpectedly increased viral pathogenicity or oncogenicity, similarly to the deletion of the entire Mid-cluster region. More importantly, our data further confirm that miR-M11-5p, the miR-M11-derived mature miRNA, targets the viral oncogene meq and suppresses its expression in GaHV2 infection. We report here that members of the Mid-clustered miRNAs, miR-M31-3p and miR-M11-5p, potentially act either as oncogene or tumour suppressor in MD lymphomagenesis.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.

In vivo expression patterns of microRNAs of Gallid herpesvirus 2 (GaHV-2) during the virus life cycle and development of Marek's disease lymphomas.

In the past decade, a large number of microRNAs (miRNAs) have been identified in the viral genome of Gallid herpesvirus 2 (GaHV-2), which is historically known as Marek's disease virus type 1. The biological role of most GaHV-2 miRNAs remains unclear. In the present study, we have performed an overall gene expression profile of GaHV-2 miRNAs during the virus life cycle at each phase of the developing disease, a highly contagious, lymphoproliferative disorder, and neoplastic immunosuppressive disease of poultry known as the Marek's disease. According to their distinct in vivo expression patterns, the GaHV-2 miRNAs can be divided into three groups: 12 miRNAs in group I, including miR-M4-5p, displayed a typical expression pattern potentially correlated to the latent, late cytolytic, and/or the proliferative phases in the cycle of GaHV-2 pathogenesis; group II consisting of another 12 miRNAs with expression correlated to the early cytolytic and/or latent phases in GaHV-2's life cycle; while the other two miRNAs in group III showed no identical expression features. Our findings may provide meaningful clues in the search for further potential functions of viral miRNAs in GaHV-2 biology.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Complete genome sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus variant.

Following the 2006 outbreaks of the highly pathogenic porcine reproductive and respiratory syndrome, the causative agent was identified as the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). To investigate whether the HP-PRRSV variant continues circulating and accelerating evolution, we sequenced and analyzed the complete genome of the identified HP-PRRSV field strain SD16. The sequence data indicate that the HP-PRRSV variant continues to prevail and accelerate evolution, especially in the nonstructural protein.

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit.

Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3'-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

Detection of the SARS-CoV-2 D614G mutation using engineered Cas12a guide RNA.

Detection of pathogens with single-nucleotide variations is indispensable for the disease tracing, but remains technically challenging. The D614G mutation in the SARS-CoV-2 spike protein is known to markedly enhance viral infectivity but is difficult to detect. Here, we report an effective approach called "synthetic mismatch integrated crRNA guided Cas12a detection" (symRNA-Cas12a) to detect the D614 and G614 variants effectively. Using this method, we systemically screened a pool of crRNAs that contain all the possible nucleotide substitutions covering the -2 to +2 positions around the mutation and identify one crRNA that can efficiently increase the detection specificity by 13-fold over the ancestral crRNA. With this selected crRNA, the symRNA-Cas12a assay can detect as low as 10 copies of synthetic mutant RNA and the results are confirmed to be accurate by Sanger sequencing. Overall, we have developed the symRNA-Cas12a method to specifically, sensitively and rapidly detect the SARS-CoV-2 D614G mutation.

Comparison of gene disruption induced by cytosine base editing-mediated iSTOP with CRISPR/Cas9-mediated frameshift.

OBJECTIVES: Recently developed CRISPR-dependent cytosine base editor (CBE), converting four codons (CAA, CAG, CGA and TGG) into stop codons without DNA double-strand breaks (DSB), serves as an efficient gene disruption strategy besides uncontrollable CRISPR-mediated frameshift. However, the detailed difference of gene knockout between the two systems has not been clarified. MATERIALS AND METHODS: Here, we selected some sgRNAs with different position background, then HEK293T cells were transfected with CBE/Cas9 plasmids together with sgRNAs. GFP-positive cells were harvested by fluorescence-activated cell sorting (FACS) 48 hours after transfection. Genomic DNA was collected for deep sequencing to analyse editing efficiency and genotype. RNA and protein were extracted to analyse gene mRNA level using qPCR analysis and Western blot. RESULTS: Here, we compared the gene disruption by CBE-mediated iSTOP with CRISPR/Cas9-mediated frameshift. We found BE-mediated gene knockout yielded fewer genotypes. BE-mediated gene editing precisely achieved silencing of two neighbouring genes, while CRISPR/Cas9 may delete the large fragment between two target sites. All of three stop codons could efficiently disrupt the target genes. It is worth notifying, Cas9-mediated gene knockout showed a more impact on neighbouring genes mRNA level than the BE editor. CONCLUSIONS: Our results reveal the differences between the two gene knockout strategies and provide useful information for choosing the appropriate gene disruption strategy.

A Cas-embedding strategy for minimizing off-target effects of DNA base editors.

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.

MeCas12a, a Highly Sensitive and Specific System for COVID-19 Detection.

Cas12a-based systems, which detect specific nucleic acids via collateral cleavage of reporter DNA, display huge potentials for rapid diagnosis of infectious diseases. Here, the Manganese-enhanced Cas12a (MeCas12a) system is described, where manganese is used to increase the detection sensitivity up to 13-fold, enabling the detection of target RNAs as low as five copies. MeCas12a is also highly specific, and is able to distinguish between single nucleotide polymorphisms (SNPs) differing by a single nucleotide. MeCas12a can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples and distinguish between SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in simulated samples, thus offering an attractive alternative to other methods for the diagnosis of infectious diseases including COVID-19 and MERS.

CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus.

African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.

Marek's disease virus type 1 encoded analog of miR-155 promotes proliferation of chicken embryo fibroblast and DF-1 cells by targeting hnRNPAB.

Marek's disease virus type 1 (MDV-1) is a representative oncogenic Alpha herpesvirus that causes an immunosuppressive and neoplastic lymphoproliferative avian disease, namely Marek's disease (MD). The rapid-onset T-cell lymphoma in chickens induced by MDV-1 has been historically regarded as an ideal natural model for herpesvirus-related cancer research. As a viral analog of cellular miR-155, the MDV-1-encoded miR-M4-5p has been shown to be crucial for the virally-induced MD tumorigenesis. Our previous studies demonstrated that miR-M4-5p induces an over-expression of oncogene c-Myc by targeting LTBP1 and suppressing the TGF-ß signaling pathway during MDV-1 infection. We have now further identified the chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) as a new cellular biological target for miR-M4-5p. Suppression of hnRNPAB expression mediated by miR-M4-5p promotes the proliferation, but not the apoptosis, of both primary chicken embryo fibroblasts (CEFs) and transformed chicken fibroblast DF-1 cell line. HnRNPAB is a member of the hnRNP family of proteins that play important roles in normal biological processes as well as cancer development. Our data suggests that the recognition and down-regulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas.

Dynamic Changes in the Splenic Transcriptome of Chickens during the Early Infection and Progress of Marek's Disease.

Gallid alphaherpesvirus 2 (GaHV2) is an oncogenic avian herpesvirus inducing Marek's disease (MD) and rapid-onset T-cell lymphomas. To reveal molecular events in MD pathogenesis and tumorigenesis, the dynamic splenic transcriptome of GaHV2-infected chickens during early infection and pathogenic phases has been determined utilizing RNA-seq. Based on the significant differentially expressed genes (DEGs), analysis of gene ontology, KEGG pathway and protein-protein interaction network has demonstrated that the molecular events happening during GaHV2 infection are highly relevant to the disease course. In the 'Cornell Model' description of MD, innate immune responses and inflammatory responses were established at early cytolytic phase but persisted until lymphoma formation. Humoral immunity in contrast began to play a role firstly in the intestinal system and started at late cytolytic phase. Neurological damage caused by GaHV2 is first seen in early cytolytic phase and is then sustained throughout the following phases over a long time period. During the proliferative phase many pathways associated with transcription and/or translation were significantly enriched, reflecting the cell transformation and lymphoma formation. Our work provides an overall view of host responses to GaHV2 infection and offers a meaningful basis for further studies of MD biology.

Genetic characterization and serological prevalence of swine hepatitis E virus in Shandong province, China.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified into four major genotypes (1 to 4) and swine is the main natural reservoir for genotypes 3 and 4. In this study, a total of 106 bile samples from a slaughterhouse in the Shandong province of China were tested for the partial ORF2 gene of HEV by RT-nPCR to determine the virus genotypes, and two indirect ELISA were developed for the detection of swine HEV specific IgM and IgG antibodies in 980 serum samples from 24 farms, in order to investigate the seroprevalence. Thirty-two out of 106 (30.2%) bile samples were positive for HEV and a high degree of partial ORF2 sequence similarity (86.8-100%) was observed among 20 samples. The viral sequences belonged to genotype 4, subtypes 4a and 4d. One complete genome sequence of a subtype 4d HEV was further determined and characterized. The seroprevalence of HEV IgG and IgM antibodies was 100% (24/24) and 41.7% (10/24) for herds, and 66.4% (651/980) and 1.6% (16/980) for the individual pigs, respectively. These results suggested a high prevalence of genotype 4 of swine HEV infection both in swine farms and at the slaughterhouse in Shandong province, which further raise public-health concerns for zoonosis and pork safety.