Drosophila Polycistronic tRNA-gRNA (PTG) Expression pattB Vector
Our Drosophila polycistronic tRNA-gRNA expression pattB vector is highly effective in generating transgenic flies that can express multiple guide RNAs (gRNAs). This system utilizes the bacteriophage φC31 integrase-mediated recombination for efficient, targeted gRNA insertion.
The attB vector system consists of two vectors, both engineered as E.coli plasmids. One vector, referred to as the attB vector or the φC31 donor vector, carries the attB site and gRNAs of interest. The other vector, referred to as the helper plasmid, encodes the φC31 integrase. When the attB and the φC31 helper plasmids are co-injected into cells containing attP landing sites, φC31 integrase mediates recombination between attB and attP sites, resulting in the linearization and integration of the attB vector into the host genome. Alternatively, the donor vector can be injected into cells from a Drosophila φC31 integrase-expressing line.
The bacteriophage φC31 encodes an integrase that mediates efficient, sequence-specific recombination between phage attachment sites (called attP) and bacterial attachment sites (called attB). In contrast to transposon-based systems, such as P-element-mediated transposition, φC31-mediated insertion is irreversible. Integration of attB into an attP position creates hybrid sites (called attL and attR), which are refractory to the φC31 integrase. Additionally, φC31-based insertion is site-specific, generally occurring only at attP sites, and not elsewhere in the genome. For this reason, the attB vector system is designed to be used with Drosophila lines carrying attP “landing sites” within their genome.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has greatly facilitated inactivation of genes in vitro and in vivo in a wide range of organisms. In this genome-editing system, the Cas9 enzyme forms a complex with a guide RNA (gRNA, which provides targeting specificity through direct interaction with homologous 18-22nt target sequences in the genome. Hybridization of the gRNA to the target site localizes Cas9, which then cuts the target site in the genome. Cas9 screens the genome and cleaves within sequences complementary to the gRNA, provided they are immediately followed by the protospacer adjacent motif (PAM) NGG. Double strand breaks are then repaired via homologous recombination or non-homologous end-joining, resulting in indels (insertion or deletion of bases in the genome) of variable length. Utilizing the CRISPR/Cas9 system in Drosophila allows the rapid generation of knockout lines by simply delivering either an all-in-one vector (a single vector expressing both Cas9 and gRNA) or separate vectors for driving Cas9 and gRNA expression, respectively.
Multiplex genome editing (MGE) is an important application of the CRISPR-Cas9 system, requiring the simultaneous expression of multiple gRNAs. To achieve effective multiplexed gene editing capability with the CRISPR/Cas9 system in Drosophila, VectorBuilder has developed the polycistronic tRNA-gRNA (PTG) expression pattB vector. In this vector, the U6-3 promoter drives the transcription of Drosophila glycine tRNA, which in turn drives the transcription of the first gRNA. The subsequently added gRNAs are each driven by rice glycine tRNA for the simultaneous production and liberation of numerous gRNAs. The transcription termination of the gRNA complex is under the control of a single terminator. Additionally, the vermillion gene on the pattB vector encodes eye color and acts as a marker for the identification of transgenic flies which have undergone successful genetic recombination. Coinjection this vector with the helper plasmid encoding φC31 integrase (or into an integrase-expressing line) and the vector encoding Cas9 into Drosophila early embryos may generate stable lines with heritable gene knockout.
For further information about this vector system, please refer to the papers below.
|Proc Natl Acad Sci U S A. 97:5995 (2000)|
Proc Natl Acad Sci U S A. 97:5995 (1998)
|Description of the φC31 integrase system|
|Proc Natl Acad Sci U S A. 104:3312-7 (2007)||Generation of φC31-based transgenic Drosophila|
|Science. 339:819-23 (2013)||Description of genome editing using the CRISPR/Cas9 system|
|Nat Methods. 13:852-4 (2016)||Introduction of the glycine tRNA-processing system based CRISPR/Cas9 multiplex gene editing tool used in Drosophila|
Our vector has been optimized to enable MGE using φC31 integrase-mediated site-specific insertion of multiple gRNAs into the Drosophila genome.
Site-specific insertion: φC31-based insertion is site-specific, generally occurring only at attP sites. This reduces the risk of disrupting endogenous genes or having insertion site position that affects transgene expression.
High efficiency: Achieving germ-line transgenesis using φC31 integrase vectors is more efficient than P-element based systems such as pUAST.
Multiplex genome editing capability: This vector contains 4 tRNA-gRNA units, which enables the pattB vector to make multiplex genome editing in Drosophila.
Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.
Requires attP insertion site: The generation of transgenic Drosophila using the pattB vector requires the use of specialized host lines carrying attP “landing sites” in their genome.
U6-3: Pol lll promoter. Drives strong expression level of small RNAs in Drosophila melanogaster.
Drosophila tRNAGly: Drosophila pre-tRNAGly gene. It is used for RNase P and Z recognition and cleavage. It also liberates multiple functional sgRNAs from a single precursor transcript in the nucleus.
Rice tRNAGly Rice pre-tRNAGly gene. It is used for RNase P and Z recognition and cleavage. It also liberates multiple functional sgRNAs from a single precursor transcript in the nucleus.
gRNA: Guide RNA compatible with the Cas9 variant being used.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
Vermilion: A selectable marker gene for Drosophila transformation. This gene encodes the enzyme required for brown eye pigment synthesis in Drosophila.
attB site: The bacterial attachment site, attB, recognized by the bacteriophage φC31 serine integrase. φC31 integrase can catalyze site-specific integration of attB-containing plasmids into attP-containing docking or landing sites that have been introduced into host genomes.