MULTIPLEXED SIV-SPECIFIC PAIRED RNA-GUIDED CAS9 NICKASES INACTIVATE PROVIRAL DNA

2021 ◽  
Author(s):  
Lisa M. Smith ◽  
Jason T. Ladner ◽  
Vida L. Hodara ◽  
Laura M. Parodi ◽  
R. Alan Harris ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rhesus Macaque ◽  
Genome Sequencing ◽  
Lymphoid Cells ◽  
Whole Genome ◽  
Dna Breaks ◽  
Proviral Dna ◽  
Cas9 Nuclease ◽  
Virus Suppression

Human and simian immunodeficiency virus infections establish lifelong reservoir of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactive integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells co-transfected with SIV provirus and plasmids coding for RGNus identified SIV LTR, TAR, and RSS regions as the most effective at virus suppression; RGNi targeting these same regions inhibited virus production significantly. Multiplex plasmids that co-expressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations making these tools a desirable prospect for moving into animal studies. Importance: Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease, or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole genome sequencing demonstrated effective SIV sequence mutation and inactivation, and absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

scholarly journals SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

scholarly journals The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1038 ◽  
Author(s):  
Jennifer H. Kopanke ◽  
Justin S. Lee ◽  
Mark D. Stenglein ◽  
Christie E. Mayo
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Stability ◽  
Bluetongue Virus ◽  
Genomic Structure ◽  
Field Isolate ◽  
Viral Population ◽  
Whole Genome ◽  
Single Nucleotide Variants

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

scholarly journals Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella

2016 ◽  
Vol 60 (9) ◽  
pp. 5515-5520 ◽  
Author(s):  
Patrick F. McDermott ◽  
Gregory H. Tyson ◽  
Claudine Kabera ◽  
Yuansha Chen ◽  
Cong Li ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
The United States ◽  
Whole Genome ◽  
Content Type ◽  
Structural Resistance ◽  
Retail Meat

ABSTRACTLaboratory-basedin vitroantimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidalSalmonellaand correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred fortySalmonellaof 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n =59) in the 104 human isolates than in the 536 retail meat isolates (n =36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1andblaSHV2a) in retail meat isolates ofSalmonellain the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidalSalmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.

scholarly journals Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

2015 ◽  
Vol 25 (3) ◽  
pp. 426-434 ◽  
Author(s):  
Brock A. Peters ◽  
Bahram G. Kermani ◽  
Oleg Alferov ◽  
Misha R. Agarwal ◽  
Mark A. McElwain ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
De Novo ◽  
Whole Genome ◽  
De Novo Mutations ◽  
Single Base

scholarly journals Analysis of Treponema pallidum Strains From China Using Improved Methods for Whole-Genome Sequencing From Primary Syphilis Chancres

2020 ◽  
Author(s):  
Wentao Chen ◽  
David Šmajs ◽  
Yongfei Hu ◽  
Wujian Ke ◽  
Petra Pospíšilová ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Treponema Pallidum ◽  
Storage Conditions ◽  
In Vitro Cultivation ◽  
Whole Genome ◽  
Primary Syphilis ◽  
Improved Methods

Abstract Background Whole-genome sequencing (WGS) of Treponema pallidum subspecies pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples. Methods We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. Results By combining parallel, pooled whole-genome amplification with hybrid selection, we generated high-quality genomes from 4 of 8 chancre-swab samples and 2 of 2 rabbit-passaged isolates, all subjected to challenging storage conditions. Conclusions This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.

scholarly journals Whole-Genome Sequencing of a Brucella melitensis Strain (BMWS93) Isolated from a Bank Clerk and Exhibiting Complete Resistance to Rifampin

2019 ◽  
Vol 8 (33) ◽  
Author(s):  
Zhi-guo Liu ◽  
Xiao-an Cao ◽  
Miao Wang ◽  
Dong-ri Piao ◽  
Hong-yan Zhao ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Inner Mongolia ◽  
Brucella Melitensis ◽  
Public Health Problem ◽  
Human Brucellosis ◽  
Whole Genome ◽  
Zone Of Inhibition ◽  
Content Type

Human brucellosis has become the most severe public health problem in the Ulanqab region of Inner Mongolia, China. Brucella melitensis BMWS93 was obtained from a blood sample taken from a bank clerk in the Ulanqab region of Inner Mongolia, China, and antimicrobial susceptibility testing in vitro showed no zone of inhibition, which confirmed resistance to rifampin. Therefore, whole-genome sequencing of this isolate was performed to better understand the mechanism of this resistance.

scholarly journals Genotyping Study of Salmonella 4,[5],12:i:- Monophasic Variant of Serovar Typhimurium and Characterization of the Second-Phase Flagellar Deletion by Whole Genome Sequencing

2020 ◽  
Vol 8 (12) ◽  
pp. 2049
Author(s):  
Ainhoa Arrieta-Gisasola ◽  
Aitor Atxaerandio-Landa ◽  
Victoria Garrido ◽  
María Jesús Grilló ◽  
Ilargi Martínez-Ballesteros ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enteritidis ◽  
Genomic Region ◽  
Second Phase ◽  
Whole Genome ◽  
Serovar Typhimurium ◽  
Selection Of

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

scholarly journals Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like

2017 ◽  
Author(s):  
Zhiting Wei ◽  
Funan He ◽  
Guohui Chuai ◽  
Hanhui Ma ◽  
Zhixi Su ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cas9 Nuclease ◽  
Mutation Pattern ◽  
And Control ◽  
Selection Of

To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

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