Drosophila Cas9 Expression pUASTB Vector (UAS-Hsp70 promoter)
Our Drosophila Cas9 expression pUASTB vector system has the capability to utilize either the Drosophila P-element transposon system (like pUAST) or the bacteriophage φC31 integration system (like pUASTattB) for Cas9 insertion into the genome. To facilitate this flexibility, the Cas9 gene is cloned in a region bracketed by two P-element terminal repeats and near an attB recombination site. This system also incorporates a strong Gal4-inducible promoter to regulate Cas9 gene expression.
The 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 18-22 nt 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.
To utilize P transposon-mediated insertion, the pUASTB plasmid and a P transposase-expressing helper plasmid are co-introduced into host cells or embryos. As a result, the transposase produced from the helper plasmid recognizes the two P-element terminal repeats on the pUASTB plasmid, and inserts the flanked region including the terminal repeats into the host genome. P transposase-mediated insertion occurs without any significant bias with respect to insertion site sequence.
To utilize φC31 integrase-mediated insertion, the pUASTB plasmid and a φC31 integrase-expressing helper plasmid are co-introduced into host cells or embryos containing attP landing sites. The φC31 integrase mediates irreversible recombination between attB and attP sites, resulting in the linearization and integration of the pUASTB vector into the host genome.
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.
In this pUASTB system, Cas9 gene is cloned downstream of an engineered, inducible promoter consisting of five tandemly arrayed GAL4 binding sites (5xUAS) and the hsp70 TATA box promoter. This GAL4/UAS system is designed to direct selective, GAL4-dependent expression of the Cas9 gene. The GAL4 protein activates gene transcription upon binding to the UAS sites upstream of Cas9. Therefore, in the absence of GAL4 expression the Cas9 gene remains silent, but introduction of GAL4 by crossing to a GAL4-expressing Drosophila line results in transcriptional activation. The GAL4 binding sites are fused to a heat shock protein hsp70 TATA box promoter. Incubation at 37℃ activates the promoter and subsequent Cas9 expression.
Additionally, the mini white gene on the pUASTB vector encodes eye color and acts as a marker for the identification of transgenic flies which have undergone successful genetic recombination of the transgene. PCR or other molecular methods can also be used to identify transgenic cells or animals.
For further information about this vector system, please refer to the papers below.
|Methods Mol Biol. 420:61 (2008)||The use of P element transposons to generate transgenic flies|
|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|
|Methods Mol Biol. 2540:135-156 (2022)||CRISPR-mediated genome editing in Drosophila|
Our Drosophila Cas9 expression pUASTB vectors are designed to achieve efficient P transposase-mediated or φC31 integrase-mediated Cas9 gene insertion and selective, GAL4-dependent expression of Cas9 protein. Our vectors are optimized for high copy number replication in E. coli and high-efficiency transgenesis of Drosophila lines.
High-level expression: The 5×UAS/mini_Hsp70 promoter can drive strong expression of the gene of interest in its induced state.
Selective expression: In the absence of GAL4, transcription of the gene of interest should be very low or silent, while in the presence of GAL4, high level of gene transcription is achieved.
High efficiency if using φC31 integrase: Achieving germ-line transgenesis using φC31 integrase vectors is more efficient than P-element based systems such as pUAST.
Random genomic insertion if using P transposase: The random integration of P-elements can make it difficult to map insertion sites, and genomic position can affect transgene expression. Additionally, transgene insertion into genes or regulatory elements within the genome can affect endogenous genes.
Moderate efficiency if using P transposase: Achieving germ-line transgenesis using P-element vectors is generally less efficient than φC31 integrase-mediated systems such as pUASTattB.
Requires attP insertion site if using φC31 integrase: The generation of transgenic Drosophila using the pUASTattB vector requires the use of specialized host lines carrying attP “landing sites” in their genome.
Potentially leaky expression: In some cases, low-level expression of the gene of interest can occur in the absence of GAL4.
Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.
P-element 3’ end: Right terminal repeat, or 3' terminal repeat, of the P-element. When a DNA sequence is flanked by the 3’ and 5’ P-element terminal repeats, the P transposase can recognize them and insert the flanked region into the host genome.
5×UAS/mini_Hsp70: The Drosophila melanogaster heat shock protein 70 (Hsp70) minimal promoter fused with five tandem galactose upstream activating sequences (5×UAS). This is a strong promoter, tightly inducible by GAL4.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
Cas9: a CRISPR-associated endonuclease that cuts DNA at a location specified by gRNA.
SV40 terminator: Simian virus 40 transcriptional terminator. Contains the SV40 small T intron and the SV40 early polyadenylation signal.
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.
mini-white: A variant of the Drosophila white gene. The mini-white gene is a dominant marker for adult fruit fly eye color, which can be used as a reporter to identify transgenic events in a white mutant background.
P-element 5’ end: Left terminal repeat, or 5' terminal repeat, of the P-element. When a DNA sequence is flanked by the 3’ and 5’ P-element terminal repeats, the P transposase can recognize them and insert the flanked region into the host genome.
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.