Drosophila Gene Targeting Donor Vector
Our Drosophila gene targeting donor vectors are designed to achieve highly efficient delivery of exogenous donor templates at genomic sites of interest based on homologous recombination. This technique allows targeted base changes, such as point mutations, or large sequence alterations such as fragment knockin.
The clustered regularly interspersed 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 18-22 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.
Homologous recombination is an essential pathway for DNA double-strand break (DSB) repair introduced by Cas9. DSBs can be repaired by homology-directed repair (HDR) using exogenous donor DNA template, which is co-introduced into cells with the CRISPR/Cas9 components. This can result in replacement of the target genomic DNA sequence with the sequence from the donor vector containing desired mutation(s) or fragments to be knocked in.
The donor vector contains the desired insertion sequence flanked by upstream and downstream homology arms. Within the homology arm-bracketed region, a loxP site-flanked fluorescent marker (DsRed) is present. The lengths of the homologous arms are adjusted depending upon the size of the desired edit, with longer insertions requiring longer arms. Users can paste their donor sequence immediately next to the sequence of the 5’ homology arm. Additionally, an attP sequence is cloned for positioning the attP landing site in the host. This produces a transgenic Drosophila embryo that will be receptive to φC31 integrase-mediated site-specific genomic recombination.
Efficient HDR targeting also requires the DSB introduced by Cas9 to be located within proximity of the target site of insertion, ideally within 10-15 bp of the homologous arms. Additionally, when designing donor vectors for HDR, it is critical to either exclude or inactivate any protospacer adjacent motif (PAM) sequences in the donor template, when present, to prevent Cas9 from disrupting the donor template or the edited genomic locus after HDR.
For further information about this vector system, please refer to the papers below.
|Methods Mol Biol. 420:155-74 (2008)|
Science. 288:2013-8 (2000)
|Homologous recombination in Drosophila|
|Biotechniques. 59:201 (2015)||CRISPR/HDR-mediated knockin of large DNA fragments|
|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|
Our gene targeting donor vectors are designed to achieve highly efficient HDR-mediated insertion of reporters, fluorescent tags or other desired sequences at genomic target sites. The donor vector is designed with the desired insertion sequence flanked by target site specific upstream and downstream homologous sequences to facilitate efficient recombination following CRISPR-generated DSBs.
Precise changes: Delivering exogenous repair templates in the form of gene targeting donor vectors enables HDR-mediated introduction of precise sequence changes at the genomic target sites of interest.
Low efficiency: Upon Cas9-induced cleavage of DNA target sites, HDR-mediated repair of the cleaved sites occurs at a much lower frequency than NHEJ-mediated repair. As a result, CRISPR/Cas9 targeting in the presence of an exogenous donor template will give rise to a mixed population of cells, some repaired by the NHEJ pathway and others repaired by the HDR pathway. Therefore, careful screening of the resultant cell population is essential to isolate clones containing the desired HDR-edited sequence. For obtaining cells with homologous alleles altered in the same way, additional round(s) of knockin screening are often needed.
PAM requirement: CRISPR/Cas9 target sites must contain an NGG sequence, known as a PAM sequence, located on the immediate 3’ end of the gRNA recognition sequence. The target must contain a PAM sequence, and any PAM sequences in the donor vector must be inactivated.
Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.
5’-Homology Arm: Homology sequence immediately upstream of the target site of insertion.
attP: φC31 attP landing site. It allows integration of an attB-containing plasmid into the attP site following HDR.
LoxP: Recognition site of Cre recombinase.
3×P3-DsRed: DsRed fluorescent marker driven by 3×3 promoter. It is used for identification of genetically engineered fly lines.
3’-Homology Arm: Homology sequence immediately downstream of the target site of insertion.
SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
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.