Drosophila Cas9 Expression pUAST Vector (UAS-Hsp70 promoter)
Our Drosophila Cas9 expression pUAST vector is highly effective in generating transgenic flies that can express Cas9 protein. This vector combines Cas9 expression for CRISPR gene editing and the P-element-based GAL4 regulated gene expression (pUAST) system.
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.
This pUAST system consists of two primary elements: (1) P-element terminal repeats for genome integration and (2) a user-defined promoter upstream of the GOI (Cas9). Genomic integration typically requires two vectors: one vector, referred to as the pUAST plasmid, contains two P-element terminal repeats bracketing the region/gene to be transposed; the other vector, referred to as the helper plasmid or transposase plasmid, encodes the P transposase. When the pUAST and the transposase plasmid are co-injected into target cells, the transposase produced from the helper plasmid recognizes the two P-element terminal repeats on the pUAST plasmid and inserts the flanked region including the terminal repeats into the host genome. The P transposase will only be expressed for a short time, and with loss of the helper plasmid, the integration of the transposon in the host genome becomes permanent. Alternatively, the pUAST plasmid can be injected into cells from a Drosophila P transposase-expressing line. Insertion occurs without any significant bias with respect to insertion site sequence. The P-element is a class-II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind (in contrast, class-I transposons move in a copy-and-paste manner.) The transposition creates 8 bp direct repeats at the integration site in the genome.
This GAL4/UAS system is designed to direct selective, GAL4-dependent expression of the Cas9 gene. The GAL4 protein activates transcription upon binding to the UAS sites upstream of the Cas9 gene. 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. Use of Drosophila lines with GAL4 under the control of tissue-specific promoters allows for selective Cas9 expression.
In this Cas9 expression pUAST vector, a Cas9 gene is cloned downstream of an engineered, inducible promoter consisting of five tandemly arrayed GAL4 binding sites (5xUAS) and the heat shock protein hsp70 TATA box promoter. Incubation at 37℃ activates the promoter and subsequent Cas9 expression. Additionally, the mini white gene on the pUAST vector encodes eye color and acts as a marker for the identification of transgenic flies which have undergone successful transposition of Cas9. 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.
|Development. 118:401 (1993)||The use of P element transposons to generate transgenic flies|
|Methods Mol Biol. 420:61 (2008)||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 pUAST vectors are designed to achieve efficient P transposase-mediated Cas9 gene insertion and selective GAL4-dependent expression of the 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.
Random genomic insertion: 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: Achieving germ-line transgenesis using P-element vectors is generally less efficient than φC31 integrase-mediated systems such as pUASTattB.
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.
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.