Prime editing is a CRISPR/Cas9-based method used to engineer precise nucleotide changes without DSBs. For prime editing, Cas9 nickase is fused with an engineered reverse transcriptase (RT) domain, together referred to as prime editor 2 (PE2). Prime editing also uses a modified guide RNA, called a prime editing guide RNA (pegRNA), which contains the intended edit and short regions of flanking homology sequence. The pegRNA directs PE2 to the target location, where it causes a single strand nick and anneals a portion of the pegRNA to the exposed genome. The RT domain then transcribes the edit from the pegRNA into the genome. Co-expressing a nicking sgRNA with the pegRNA (referred to as the PE3 system) can increase editing efficiency, but also may resulted in indels at the target site.
- edit somatic and germline cells
- generate edits by transgenic crossing
- So far only been used to edit or insert small regions (up to ~100 bp)
To start making edits with the PE2 system:
- obtain fly stocks from BDSC for expression of the PE2 enzyme (90968, 90971, 90974, 90977, 91349, 91350)
- clone your desired pegRNA into pCFD3-NS (Addgene 149545, DGRC 1528)
- generate transgenic pegRNA flies
- set up transgenic cross to make edit
To start making edits with the PE3 system:
- obtain fly stocks from BDSC for expression of the PE2 enzyme (90968, 90971, 90974, 90977, 91349, 91350)
- clone your desired pegRNA and sgRNA into pCFD5-NS (Addgene 149546, DGRC 1529)
- generate transgenic pegRNA-sgRNA flies
- set up transgenic cross to make edit
For a detailed description of Prime editing in flies see : Bosch JA, Birchak G, Perrimon N. Precise genome engineering in Drosophila using prime editing. Proc Natl Acad Sci U S A. 2021 Jan 5;118(1):e2021996118. doi: 10.1073/pnas.2021996118. PMID: 33443210; PMCID: PMC7817132.
Prime editing plasmids
| plasmid | Addgene | DGRC | purpose |
| pNos-PE2-attB | 149549 | 1525 | Expression of PE2 enzyme under control of the germ cell specific nanos promoter. Can be used to generate transgenic flies with vermillion+ selection. |
| pUAS-PE2-attB | 149550 | 1527 | Expression of PE2 enzyme under control of the Gal4-regulated UAS promoter. Can be used to generate transgenic flies with white+ selection. |
| pEntr_PE2 | 149548 | 1526 | Gateway entry clone with PE2 enzyme (with stop codon). |
| pCFD3-NS | 149545 | 1528 | Expression of single pegRNA under control of Drosophila U6:3 promoter. NS stands for "No Scaffold". Can be used to generate transgenic flies with vermillion+ selection. |
| pCFD5-NS | 149546 | 1529 | Expression of pegRNA(s) and sgRNA(s) under control of Drosophila U6:3 promoter for PE3 system. NS stands for "No Scaffold". Can be used to generate transgenic flies with vermillion+ selection. |
Prime editing fly stocks
| BDSC ID | Genotype | Purpose |
| 90968 | w[*]; P{y[+t7.7] w[+mC]=UAS-PE2}attP2/TM6B, Tb[1] | Expresses the Prime Editor 2 enzyme complex under UAS control |
| 90969 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE-e[G111X]}attP40/CyO | Ubiquitously expresses a pegRNA targeting the ebony gene to introduce the G111X mutation. Positive control for the Prime Editing system |
| 90970 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE-f[D111X]}attP40/CyO | Ubiquitously expresses a pegRNA targeting the forked gene to introduce the D111X mutation. Positive control for the Prime Editing system |
| 90971 | w[*]; P{y[+t7.7] w[+mC]=UAS-PE2}attP40 | Expresses the Prime Editor 2 enzyme complex under UAS control |
| 90973 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE-w[A134X]}attP40/CyO | Ubiquitously expresses a pegRNA targeting the white gene to introduce the A134X mutation |
| 90974 | w[*]; P{y[+t7.7] w[+mC]=UAS-PE2}attP40; P{w[+mC]=tubP-GAL4}LL7/TM6B, Tb[1] | Expresses the Prime Editor 2 enzyme complex under UAS control combined with ubiquitous GAL4 |
| 90975 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE3-e[G111X]}attP40 | Ubiquitously expresses both a pegRNA and sgRNA targeting the ebony gene to introduce the G111X mutation. Positive control for the Prime Editing 3 system |
| 90976 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE3-w[A134X]}attP40/CyO | Ubiquitously expresses both a pegRNA and sgRNA targeting the white gene to introduce the A134X mutation. Positive control for the Prime Editing 3 system |
| 90977 | w[*]; P{w[+mC]=Act5C-GAL4}25FO1/CyO; P{y[+t7.7] w[+mC]=UAS-PE2}attP2 | Expresses the Prime Editor 2 enzyme complex under UAS control combined with ubiquitous GAL4 |
| 90978 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=U6-PE3-f[D111X]}attP40/CyO | Ubiquitously expresses both a pegRNA and sgRNA targeting the forked gene to introduce the D111X mutation. Positive control for the Prime Editing 3 system |
| 91349 | w[*]; P{w[+mC]=GAL4-nos.NGT}40, P{y[+t7.7] w[+mC]=UAS-PE2}attP40/CyO | Expresses the Prime Editor 2 enzyme complex under UAS control combined with germ cell GAL4 |
| 91350 | w[*]; P{y[+t7.7] w[+mC]=UAS-PE2}attP2, P{w[+mC]=GAL4::VP16-nos.UTR}CG6325[MVD1] | Expresses the Prime Editor 2 enzyme complex under UAS control combined with germ cell GAL4 |