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Drosophila Cas9 Expression pUASTB Vector (User-Defined promoter)

Overview

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 user-defined promoter to achieve ubiquitous, tissue-specific or inducible Cas9 protein 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, users can either paste sequences for their own promoter or select a promoter from our database. Our Drosophila promoter database offers the following promoter choices: ubiquitous promoters including actin 5C, polyubiquitin and alpha-1 tubulin; tissue-specific promoters such as Rh2 for driving GOI expression specifically in Drosophila ocelli; and inducible promoters such as Mtn and DmHsp70 for achieving expression in the presence of Cu+ and in response to heat stress, respectively. 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 integration 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.

References Topic
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

Highlights

Our Drosophila Cas9 expression pUASTB vectors are designed to achieve efficient P transposase-mediated or φC31 integrase-mediated Cas9 gene insertion. Our vectors are optimized for high copy number replication in E. coli and high-efficiency transgenesis of Drosophila lines. The user-defined promoter version of this vector allows users to select a ubiquitous, tissue-specific, or inducible promoter for driving Cas9 gene expression.

Advantages

Flexibility: The user-defined promoter version of the pUAST vector allows users to select a ubiquitous, tissue-specific or inducible promoter for driving their GOI depending upon their experimental goal.

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.

Disadvantages

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.

Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.

Key components

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.

Promoter: A DNA sequence upstream of a gene to which proteins bind to initiate transcription of that gene.

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.

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