epegRNA Pooled Libraries
(Pooled Library #227706, #227707, #227708)

• Purpose:

  The StopPR epegRNA library (227706) contains engineered prime editing guide RNA (epegRNA) constructs, which include epegRNA components (guide, primer binding site, and reverse transcriptase template). This library targets premature stop codons to essential genes, and can therefore be used to assess prime editing-induced dropout phenotypes in a pooled format across thousands of endogenous target sites.

  The +5 G>H Self-targeting epegRNA library (227707) contains self-targeting epegRNA constructs, which include epegRNA components (guide, primer binding site, and reverse transcriptase template) as well as an integrated target site. This library can therefore be used to assess prime editing efficiency in a pooled format across hundreds of target sites.

  The Tiled-Edits Self-targeting epegRNA library (227708) contains self-targeting epegRNA constructs, which include epegRNA components (guide, primer binding site, and reverse transcriptase template) as well as an integrated target site. This library can therefore be used to assess prime editing efficiency in a pooled format across five target sites.

• Vector Backbone: 227706 StopPR epegRNA library:

  • pAC026 (Plasmid #227598) - does not express Cas9

  227707 and 22708 self-targeting epegRNA libraries:

  • pAC025 (Plasmid #227597) - does not express Cas9

• Depositing Labs: Brittany Adamson
• Publication: Cirincione et al. Nat Methods. 2024 Nov 19. doi: 10.1038/s41592-024-02502-4.

Library Details

	227706	227707	227708
Species	Human	Human	Mouse
Genes targeted	1,232 essential genes across 17,061 codons, with 42,269 unique edits	640 target sites, with 3 edits per target site	113 target sites, with 1–65 edits per target site
epegRNAs per element	Up to 8 per edit	1 per edit	20 per edit
Controls	94,724 synonymous, 12,000 no edit, and 3,000 non-targeting	22 positive and 51 non-targeting control target pairs	113 negative, 5 positive
Totel number of epegRNAs	240,000	2,000 target pairs	5,960 target pairs

• Lentiviral Generation: 3rd

Library Shipping

Each library is delivered in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

• Volume: ∼15 µL
• Concentration: 50 ng/µL

Resource Information

• Protocols:
  • Pooled epegRNA Library Amplification (DOCX, 19 KB)
  • Pooled epegRNA Validation (DOCX, 20 KB)
• Depositor Data:
  • List of StopPR epegRNA Library (lAC002) Sequences (CSV, 20 MB)
  • List of Self-targeting epegRNA Library, +5 G>H (lDS004) Sequences (CSV, 306 KB)
  • List of Self-targeting epegRNA Library, Tiled Edits (lRM001) Sequences (XLSX, 407 KB)
• Scripts:
  • Code used to analyze prime editing epegRNA dropout from a screen using the stopPR epegRNA library (lAC002).
  • Code used to analyze prime editing efficiency from a self-targeting screen using the self-targeting epegRNA library, +5 G>H (lDS004).
  • Code used to analyze prime editing efficiency from a self-targeting screen using the self-targeting epegRNA library, tiled edits (lRM001).

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA
• Evrogen Limited Use Label License for FPs

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

The depositing lab originally used this library with K562 cell lines engineered to stably express the PEmax protein (composed in part with a Cas9 nickase). These cell lines were generated in this study and a previous study (PMID: 38570691 ).

Additional library details:

Number of genes/target sites

• 227706: 1,232 essential genes targeted across 17,061 codons, with 42,269 unique edits.
• 227707: 640 target sites (genomic sequences that have been integrated into oligo constructs to make a self-targeting epegRNA library) and 3 edits per target site.
• 227708: 113 target sites (genomic sequences that have been integrated into oligo constructs to make a self-targeting epegRNA library) and 1–65 edits per target site.

Number of controls

• 227706: 94,724 epegRNAs specifying edits that do not change an amino acid and thus were not designed to alter protein function (synonymous epegRNAs), 12,000 epegRNAs designed to introduce no change in sequence (epegRNA matches the unedited reference sequence; no edit epegRNAs), and 3,000 epegRNAs containing scrambled non-targeting spacer sequences (non-targeting epegRNAs).
• 227707: 22 positive control epegRNA-target pairs (targeting sites tested endogenously in the literature) and 51 non-targeting control epegRNA-target pairs (with a scrambled target site sequence).
• 227708: 113 epegRNAs encoding the unedited sequence with a primer binding site length of 7 nt and reverse transcriptase template length of 13 nt were included as negative controls, 1 for each target site. 5 previously validated epegRNA designs encoding single nucleotide variant edits in Rnf2 (1 edit), Dnmt1 (2 edits), and Hoxd13 (2 edits) were included as positive controls.

Total number of pegRNAs

• 227706: 240,000 epegRNAs. Note that 580 pairs of stop epegRNAs and spacer/codon-matched synonymous epegRNAs were removed from analysis in the manuscript, after a computational pipeline update.
• 227707: 2,000 epegRNA-target pairs.
• 227708: 5,960 epegRNA-target pairs, including 3,754 corresponding to the “Tiled edits” ZRS enhancer site tiling screen presented in the manuscript.

Please visit https://doi.org/10.1101/2024.03.25.585978 for bioRxiv preprint.