Pub Date : 2020-09-23eCollection Date: 2020-01-01DOI: 10.7150/jgen.51181
Samira Mansour, Erik Swanson, Céline Pesce, Stephen Simpson, Krystalynne Morris, W Kelley Thomas, Louis S Tisa
Frankia sp. strains CgS1, CcI156 and CgMI4 were isolated from Casuarina glauca and C. cunninghamiana nodules. Here, we report the 5.26-, 5.33- and 5.20-Mbp draft genome sequences of Frankia sp. strains CgS1, CcI156 and CgMI4, respectively. Analysis of the genome revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters.
{"title":"Draft Genome Sequences for the <i>Frankia</i> sp. strains CgS1, CcI156 and CgMI4, Nitrogen-Fixing Bacteria Isolated from <i>Casuarina</i> sp. in Egypt.","authors":"Samira Mansour, Erik Swanson, Céline Pesce, Stephen Simpson, Krystalynne Morris, W Kelley Thomas, Louis S Tisa","doi":"10.7150/jgen.51181","DOIUrl":"https://doi.org/10.7150/jgen.51181","url":null,"abstract":"<p><p><i>Frankia</i> sp. strains CgS1, CcI156 and CgMI4 were isolated from <i>Casuarina glauca</i> and C. <i>cunninghamiana</i> nodules. Here, we report the 5.26-, 5.33- and 5.20-Mbp draft genome sequences of <i>Frankia</i> sp. strains CgS1, CcI156 and CgMI4, respectively. Analysis of the genome revealed the presence of high numbers of secondary metabolic biosynthetic gene clusters.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"84-88"},"PeriodicalIF":0.0,"publicationDate":"2020-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.51181","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38468302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-08-29eCollection Date: 2020-01-01DOI: 10.7150/jgen.50468
Takashi Kitano, Choong-Gon Kim, Naruya Saitou
We sequenced the western gorilla (Gorilla gorilla) HoxA cluster region using seven fosmid clones, and found that the total tiling path sequence was 214,185 bp from the 5' non-genic region of HoxA1 to the 3' non-genic region of Evx1. We compared the nucleotide sequence with the gorilla genome sequence in the NCBI database, and the overall proportion of nucleotide difference was estimated to be 0.0005-0.0007. These estimates are lower than overall genomic polymorphism in gorillas.
{"title":"Nucleotide sequencing of the HoxA gene cluster using Gorilla fosmid clones.","authors":"Takashi Kitano, Choong-Gon Kim, Naruya Saitou","doi":"10.7150/jgen.50468","DOIUrl":"https://doi.org/10.7150/jgen.50468","url":null,"abstract":"<p><p>We sequenced the western gorilla (<i>Gorilla gorilla</i>) HoxA cluster region using seven fosmid clones, and found that the total tiling path sequence was 214,185 bp from the 5' non-genic region of HoxA1 to the 3' non-genic region of Evx1. We compared the nucleotide sequence with the gorilla genome sequence in the NCBI database, and the overall proportion of nucleotide difference was estimated to be 0.0005-0.0007. These estimates are lower than overall genomic polymorphism in gorillas.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"80-83"},"PeriodicalIF":0.0,"publicationDate":"2020-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.50468","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38481460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-07-03eCollection Date: 2020-01-01DOI: 10.7150/jgen.42790
Belen Gonzalez-Santamarina, Anne Busch, Silvia Garcia-Soto, Mostafa Y Abdel-Glil, Jörg Linde, Reinhard Fries, Diana Meemken, Helmut Hotzel, Herbert Tomaso
We report the first draft genome sequence of a Salmonella strain with plasmid-mediated resistance to colistin encoded by mcr-1 gene in Vietnam. Salmonella enterica subsp. enterica serovar Rissen was isolated from a Vietnamese pig slaughterhouse in 2013. We can confirm that mcr-1 gene is identical to the first reported mcr-1 gene of the Escherichia coli strain SHP45, isolated in 2015 from a Chinese pig. The plasmid containing this gene in the strain 19CS0416 was highly related (96.86% identity) to the plasmid (pHNSHP45) contained in this Chinese strain. Moreover, this plasmid was determined to be 100% identical to a plasmid (p13P477T-7) belonging to an Escherichia coli (13P477T) found in Hong Kong during the same year in pigs. Our results will aid in understanding the dissemination of mcr-1 gene in East Asia, dating back to as early as 2013.
{"title":"Draft genome sequence of multi-resistant <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Rissen strain 19CS0416 isolated from Vietnam reveals <i>mcr-1</i> plasmid mediated resistance to colistin already in 2013.","authors":"Belen Gonzalez-Santamarina, Anne Busch, Silvia Garcia-Soto, Mostafa Y Abdel-Glil, Jörg Linde, Reinhard Fries, Diana Meemken, Helmut Hotzel, Herbert Tomaso","doi":"10.7150/jgen.42790","DOIUrl":"https://doi.org/10.7150/jgen.42790","url":null,"abstract":"<p><p>We report the first draft genome sequence of a <i>Salmonella</i> strain with plasmid-mediated resistance to colistin encoded by <i>mcr-1</i> gene in Vietnam. <i>Salmonella enterica subsp. enterica</i> serovar Rissen was isolated from a Vietnamese pig slaughterhouse in 2013. We can confirm that <i>mcr-1</i> gene is identical to the first reported <i>mcr-1</i> gene of the <i>Escherichia coli</i> strain SHP45, isolated in 2015 from a Chinese pig. The plasmid containing this gene in the strain 19CS0416 was highly related (96.86% identity) to the plasmid (pHNSHP45) contained in this Chinese strain. Moreover, this plasmid was determined to be 100% identical to a plasmid (p13P477T-7) belonging to an <i>Escherichia coli</i> (13P477T) found in Hong Kong during the same year in pigs. Our results will aid in understanding the dissemination of <i>mcr-1</i> gene in East Asia, dating back to as early as 2013.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"76-79"},"PeriodicalIF":0.0,"publicationDate":"2020-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.42790","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38283674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-06-29eCollection Date: 2020-01-01DOI: 10.7150/jgen.48656
Anne Busch, Helmut Hotzel, Ulrich Methner
We describe a rare isolate of Salmonella enterica subsp. enterica serovar Panama with an extended-spectrum β-lactamase (ESBL) profile from a German cattle-fattening farm. Applying two next-generation sequencing methods we generated sequences of the genome as well as the plasmids; assembled the draft genome sequence of Salmonella enterica subsp. enterica serovar Panama isolate 18PM0209. Antimicrobial resistance genes, virulence-associated genes and plasmids were analyzed using bioinformatics. Occurrence of multidrug-resistant Salmonella serovars at cattle-fattening farms indicate the need of enhanced surveillance to prevent further spread of these organisms.
{"title":"Complete genome and plasmid sequences of a multidrug-resistant Salmonella enterica subsp. enterica serovar Panama isolate from a German cattle farm.","authors":"Anne Busch, Helmut Hotzel, Ulrich Methner","doi":"10.7150/jgen.48656","DOIUrl":"https://doi.org/10.7150/jgen.48656","url":null,"abstract":"<p><p>We describe a rare isolate of Salmonella enterica subsp. enterica serovar Panama with an extended-spectrum β-lactamase (ESBL) profile from a German cattle-fattening farm. Applying two next-generation sequencing methods we generated sequences of the genome as well as the plasmids; assembled the draft genome sequence of Salmonella enterica subsp. enterica serovar Panama isolate 18PM0209. Antimicrobial resistance genes, virulence-associated genes and plasmids were analyzed using bioinformatics. Occurrence of multidrug-resistant <i>Salmonella</i> serovars at cattle-fattening farms indicate the need of enhanced surveillance to prevent further spread of these organisms.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"71-75"},"PeriodicalIF":0.0,"publicationDate":"2020-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.48656","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38283673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-05-18eCollection Date: 2020-01-01DOI: 10.7150/jgen.41196
Dylan Beeber, Frédéric Jj Chain
The success of CRISPR/Cas9 gene editing applications relies on the efficiency of the single guide RNA (sgRNA) used in conjunction with the Cas9 protein. Current sgRNA design software vary in the details they provide on sgRNA sequence efficiency and usually limit organism choice to a list of developer-selected species. The crispRdesignR package aims to address these limitations by providing comprehensive sequence features of the generated sgRNAs in a single program, which allows users to predict sgRNA efficiency and design sgRNA sequences for systems that currently do not have optimized efficiency scoring methods. crispRdesignR reports extensive information on all designed sgRNA sequences with robust off-target calling and annotation and can be run in a user-friendly graphical interface. The crispRdesignR package is implemented in R and has fully editable code for specialized purposes including sgRNA design in user-provided genomes. The package is platform independent and extendable, with its source code and documentation freely available at https://github.com/dylanbeeber/crispRdesignR.
{"title":"crispRdesignR: A Versatile Guide RNA Design Package in R for CRISPR/Cas9 Applications.","authors":"Dylan Beeber, Frédéric Jj Chain","doi":"10.7150/jgen.41196","DOIUrl":"https://doi.org/10.7150/jgen.41196","url":null,"abstract":"<p><p>The success of CRISPR/Cas9 gene editing applications relies on the efficiency of the single guide RNA (sgRNA) used in conjunction with the Cas9 protein. Current sgRNA design software vary in the details they provide on sgRNA sequence efficiency and usually limit organism choice to a list of developer-selected species. The <i>crispRdesignR</i> package aims to address these limitations by providing comprehensive sequence features of the generated sgRNAs in a single program, which allows users to predict sgRNA efficiency and design sgRNA sequences for systems that currently do not have optimized efficiency scoring methods. <i>crispRdesignR</i> reports extensive information on all designed sgRNA sequences with robust off-target calling and annotation and can be run in a user-friendly graphical interface. The <i>crispRdesignR</i> package is implemented in R and has fully editable code for specialized purposes including sgRNA design in user-provided genomes. The package is platform independent and extendable, with its source code and documentation freely available at https://github.com/dylanbeeber/crispRdesignR.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"62-70"},"PeriodicalIF":0.0,"publicationDate":"2020-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.41196","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38006908","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Actinomadura sp. K4S16 (=NBRC 110471) is a producer of a novel tetronate polyether compound nonthmicin. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation and analysis of the genome sequence. The 9.6 Mb genome of Actinomadura sp. K4S16 encoded 9,004 putative ORFs, of which 7,701 were assigned with COG categories. The genome contained four type-I polyketide synthase (PKS) gene clusters, two type-II PKS gene clusters, and three nonribosomal peptide synthetase (NRPS) gene clusters. Among the type-I PKS gene (t1pks) clusters, a large t1pks cluster was annotated to be responsible for nonthmicin synthesis based on bioinformatic analyses. We also performed feeding experiments using labeled precursors and propose the biosynthetic pathway of nonthmicin.
{"title":"Draft genome sequence of <i>Actinomadura</i> sp. K4S16 and elucidation of the nonthmicin biosynthetic pathway.","authors":"Hisayuki Komaki, Enjuro Harunari, Natsuko Ichikawa, Akira Hosoyama, Moriyuki Hamada, Kannika Duangmal, Arinthip Thamchaipenet, Yasuhiro Igarashi","doi":"10.7150/jgen.44650","DOIUrl":"10.7150/jgen.44650","url":null,"abstract":"<p><p><i>Actinomadura</i> sp. K4S16 (=NBRC 110471) is a producer of a novel tetronate polyether compound nonthmicin. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation and analysis of the genome sequence. The 9.6 Mb genome of <i>Actinomadura</i> sp. K4S16 encoded 9,004 putative ORFs, of which 7,701 were assigned with COG categories. The genome contained four type-I polyketide synthase (PKS) gene clusters, two type-II PKS gene clusters, and three nonribosomal peptide synthetase (NRPS) gene clusters. Among the type-I PKS gene (<i>t1pks</i>) clusters, a large <i>t1pks</i> cluster was annotated to be responsible for nonthmicin synthesis based on bioinformatic analyses. We also performed feeding experiments using labeled precursors and propose the biosynthetic pathway of nonthmicin.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"53-61"},"PeriodicalIF":0.0,"publicationDate":"2020-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256010/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38006907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-04-22eCollection Date: 2020-01-01DOI: 10.7150/jgen.43953
Indika Senevirathna, Dinesha Jayasundara, Joshua P Lefler, Kira L Chaiboonm, Janith Warnasekara, Suneth Agampodi, Michael A Matthias, Joseph M Vinetz
Leptospirosis is an important cause of acute undifferentiated fever and complex multisystem febrile diseases in the tropics and subtropics. Understanding the evolution of Leptospira especially as related to the clinical pathogenesis of leptospirosis is facilitated by systematic comparative genomic analysis of human-infecting isolates. Here, we announce the complete genome sequences of three Leptospira strains that were isolated from blood of humans with undifferentiated fever in Sri Lanka.
{"title":"Complete Genome Sequence of <i>Leptospira interrogans</i> Strains FMAS_KW1, FMAS_KW2 and FMAS_AW1 Isolated from Leptospirosis Patients from Karawanalla and Awissawella, Sri Lanka.","authors":"Indika Senevirathna, Dinesha Jayasundara, Joshua P Lefler, Kira L Chaiboonm, Janith Warnasekara, Suneth Agampodi, Michael A Matthias, Joseph M Vinetz","doi":"10.7150/jgen.43953","DOIUrl":"https://doi.org/10.7150/jgen.43953","url":null,"abstract":"<p><p>Leptospirosis is an important cause of acute undifferentiated fever and complex multisystem febrile diseases in the tropics and subtropics. Understanding the evolution of <i>Leptospira</i> especially as related to the clinical pathogenesis of leptospirosis is facilitated by systematic comparative genomic analysis of human-infecting isolates. Here, we announce the complete genome sequences of three <i>Leptospira</i> strains that were isolated from blood of humans with undifferentiated fever in Sri Lanka.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"49-52"},"PeriodicalIF":0.0,"publicationDate":"2020-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.43953","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38006906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-04-07eCollection Date: 2020-01-01DOI: 10.7150/jgen.39147
Tomasz Szmatoła, Artur Gurgul, Igor Jasielczuk, Weiwei Fu, Katarzyna Ropka-Molik
In the present study, runs of homozygosity (ROH) detected with the use of a standard bovine 54k single nucleotide polymorphism (SNP) genotyping assay and two different ROH detection approaches, based on 50 (M1) or 15 (M2) consecutive SNPs, were compared with results of whole genome sequencing. Both microarray-based methods accurately recognised medium-sized ROH, however, it was found that M2 method seemed to better than M1 identify short ROH, but highly overestimated their number, leading to numerous false positive calls. Moreover, long ROH identified with microarray data tended to break into shorter segments in sequencing data because of the presence of regions with high heterozygosity within the ROH sequences. This may indicate, that these long ROH are formed by closely positioned shorter homozygous segments that may be of older origin or may be created by two similar but not identical haplotypes, showing minor internal recombination signs. Such finding also suggests that at least some of the results of previous studies in regard to long ROH may be biased leading to inaccurate estimations of genomes autozygosity via ROH classification into length categories.
{"title":"A detailed characteristics of bias associated with long runs of homozygosity identification based on medium density SNP microarrays.","authors":"Tomasz Szmatoła, Artur Gurgul, Igor Jasielczuk, Weiwei Fu, Katarzyna Ropka-Molik","doi":"10.7150/jgen.39147","DOIUrl":"https://doi.org/10.7150/jgen.39147","url":null,"abstract":"<p><p>In the present study, runs of homozygosity (ROH) detected with the use of a standard bovine 54k single nucleotide polymorphism (SNP) genotyping assay and two different ROH detection approaches, based on 50 (M1) or 15 (M2) consecutive SNPs, were compared with results of whole genome sequencing. Both microarray-based methods accurately recognised medium-sized ROH, however, it was found that M2 method seemed to better than M1 identify short ROH, but highly overestimated their number, leading to numerous false positive calls. Moreover, long ROH identified with microarray data tended to break into shorter segments in sequencing data because of the presence of regions with high heterozygosity within the ROH sequences. This may indicate, that these long ROH are formed by closely positioned shorter homozygous segments that may be of older origin or may be created by two similar but not identical haplotypes, showing minor internal recombination signs. Such finding also suggests that at least some of the results of previous studies in regard to long ROH may be biased leading to inaccurate estimations of genomes autozygosity via ROH classification into length categories.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"43-48"},"PeriodicalIF":0.0,"publicationDate":"2020-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.39147","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37867345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bifidobacterium species are well recognized as probiotics and colonized in various parts of the human body. Here, we report the draft genome sequences of Bifidobacterium animalis isolated from two healthy Japanese volunteers, one of which was sampled twice before and after a 10-year interval. A core genome phylogeny analysis indicated that the strains isolated from the same volunteer were closely related. This paper is the first report of multiple draft genome sequences of B. animalis independently isolated from the same individual and provides insight into the probiotic potential of a member of this species.
{"title":"Draft Genome Sequences of <i>Bifidobacterium animalis</i> Consecutively Isolated from Healthy Japanese Individuals.","authors":"Tomoya Tsukimi, Tsubasa Watabe, Kazuki Tanaka, Mitsuhiko P Sato, Haruo Suzuki, Masaru Tomita, Shinji Fukuda","doi":"10.7150/jgen.38516","DOIUrl":"https://doi.org/10.7150/jgen.38516","url":null,"abstract":"<p><p><i>Bifidobacterium</i> species are well recognized as probiotics and colonized in various parts of the human body. Here, we report the draft genome sequences of <i>Bifidobacterium animalis</i> isolated from two healthy Japanese volunteers, one of which was sampled twice before and after a 10-year interval. A core genome phylogeny analysis indicated that the strains isolated from the same volunteer were closely related. This paper is the first report of multiple draft genome sequences of <i>B. animalis</i> independently isolated from the same individual and provides insight into the probiotic potential of a member of this species.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"37-42"},"PeriodicalIF":0.0,"publicationDate":"2020-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.38516","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37867406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2020-03-01eCollection Date: 2020-01-01DOI: 10.7150/jgen.43928
Aditi Kulkarni, Wanqin Yu, Alex S Moon, Ashmita Pandey, Kathryn A Hanley, Jiannong Xu
In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.
{"title":"Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes.","authors":"Aditi Kulkarni, Wanqin Yu, Alex S Moon, Ashmita Pandey, Kathryn A Hanley, Jiannong Xu","doi":"10.7150/jgen.43928","DOIUrl":"https://doi.org/10.7150/jgen.43928","url":null,"abstract":"<p><p>In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A <i>Cas13a</i> expressing plasmid was delivered to mosquitoes by intrathoracic injection, and <i>Cas13a</i> transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized <i>in vitro</i> using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In <i>Anopheles gambiae</i>, <i>vitellogenin</i> gene was silenced by Cas13a/<i>Vg-</i>crRNA, which was accompanied by a significant reduction in egg production. In <i>Aedes aegypti</i>, the α- and δ-subunits of <i>COPI</i> genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.</p>","PeriodicalId":15834,"journal":{"name":"Journal of Genomics","volume":"8 ","pages":"30-36"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.7150/jgen.43928","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37753233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}