Scarless engineering of the Drosophila genome near any site-specific integration site

Abstract We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

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Scarless engineering of the Drosophila genome near any site-specific integration site

10.1101/2020.08.13.249656 ◽

2020 ◽

Author(s):

Siqian Feng ◽

Shan Lu ◽

Wesley B. Grueber ◽

Richard S. Mann

Keyword(s):

Integration Site ◽

Landing Site ◽

Genomic Sequences ◽

Efficient Technique ◽

Drosophila Genome ◽

Step Method ◽

Phic31 Integrase ◽

Site Specific ◽

Site Specific Integration ◽

Landing Sites

AbstractWe describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This 2-step method combines phiC31 integrase mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.

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Transcriptional Analysis of the Adeno-Associated Virus Integration Site

Journal of Virology ◽

10.1128/jvi.01754-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12512-12525 ◽

Cited By ~ 5

Author(s):

Nathalie Dutheil ◽

Els Henckaerts ◽

Erik Kohlbrenner ◽

R. Michael Linden

Keyword(s):

Transcriptional Activity ◽

Integration Site ◽

Regulatory Elements ◽

Transcriptional Analysis ◽

Start Codon ◽

Adeno Associated Virus ◽

Site Specific ◽

Complex Interactions ◽

Site Specific Integration ◽

Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

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Adeno-Associated Virus Site-Specific Integration Is Regulated by TRP-185

Journal of Virology ◽

10.1128/jvi.02014-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1990-2001 ◽

Cited By ~ 3

Author(s):

Noriaki Yamamoto ◽

Masato Suzuki ◽

Masa-aki Kawano ◽

Takamasa Inoue ◽

Ryou-u Takahashi ◽

...

Keyword(s):

Nonstructural Protein ◽

Integration Site ◽

Dna Helicase ◽

Binding Property ◽

Helicase Activity ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Dna Binding Property ◽

Aavs1 Locus

ABSTRACT Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3′→5′ DNA helicase activity.

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Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Microbiology ◽

10.1099/mic.0.26318-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2443-2453 ◽

Cited By ~ 8

Author(s):

Dylan C. Alexander ◽

David J. Devlin ◽

Duane D. Hewitt ◽

Ann C. Horan ◽

Thomas J. Hosted

Keyword(s):

Escherichia Coli ◽

Sequence Analysis ◽

Integration Site ◽

Attachment Site ◽

Bacterial Attachment ◽

Temperate Bacteriophage ◽

Site Specific ◽

Sequence Identity ◽

Site Specific Integration ◽

Chromosome Sequence

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

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