PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells
- Institute of Enzymology, Research Centre for Natural Sciences, Hungary
- ProteoScientia, Hungary
- School of Ph.D. Studies, Semmelweis University, Hungary
- Biospiral-2006, Hungary
- Institute of Biochemistry, Biological Research Centre, Hungary
Figures
Figure 1 with 1 supplement
Principle of the prime editor activity reporter (PEAR) assay.
(A) Schematic of the PEAR. The mechanism of PEAR is based on the same concept as BEAR (see Figure 1—figure supplement 1A and B), and it contains the same inactive splice site, shown in (A). PE can …
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Figure 1—source data 1
Measured values for Figure 1.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The differences between the base editor activity reporter (BEAR) and PEAR assays.
(A) Schematic representation of the BEAR. BEAR consists of a split GFP coding sequence separated by an intron, of which the 5′ splice donor site (G-GT-AAGT) is altered, resulting in an inactive …
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Figure 1—figure supplement 1—source data 1
Measured values for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 2 supplements
Prime editing with PEAR in a genomic and plasmid context.
PEs targeting BEAR sequences either located in plasmids (Plasmid) or incorporated into the genome (Cell line) were transfected into cells alongside different sgRNAs for different complementary …
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Figure 2—source data 1
Measured values for Figure 2.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Gating examples from HEK-BEAR cell lines.
Gating examples are shown from Figure 2 where cells harboring a genomically integrated copy of BEAR-mScarlet (A) or BEAR-GFP (B) (HEK-BEAR-mScarlet and HEK-BEAR-GFP cell lines, respectively) …
Figure 2—figure supplement 2
Prime editing of the BEAR-mScarlet plasmid with target 2.
(A) Scatter plot of GFP-positive cells measured either when the BEAR-GFP plasmid or the HEK-BEAR-GFP cell line (Figure 2A) was edited by PE3 with various pegRNA and secondary nicking sgRNA …
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Figure 2—figure supplement 2—source data 1
Measured values for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
PEAR enriches prime edited cells.
(A) Flow cytometry measurements of prime edited (i.e., mScarlet positive) cells in the HEK-BEAR-mScarlet cell line. The PEAR-GFP plasmid was co-transfected with PE protein-coding plasmid, two …
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Figure 3—source data 1
Measured values and detailed statistics for Figure 3.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Stable integration of PEAR plasmids.
PEAR enriched populations (sorted for GFP, day 0) of HEK293T cells from the experiments in Figure 3C were passaged for 3–4 weeks. GFP-positive cells were measured by flow cytometry to determine …
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Figure 3—figure supplement 1—source data 1
Measured values for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig3-figsupp1-data1-v1.xlsx
Figure 4
Enrichment with a PEAR reporter with no off-target activity.
(A) Potential off-target cleavage of WT-SpCas9 was assessed by GUIDE-seq on EGFP site 7. Read counts are shown for the on-target sequence only, as no off-targets were identified. On the bar charts, …
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Figure 4—source data 1
Measured values and detailed statistics for Figure 4.
- https://cdn.elifesciences.org/articles/69504/elife-69504-fig4-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | |
| Genetic reagent (H. sapiens) | HEK293.EGFP | Kulcsár et al., 2017 | ||
| Cell line (H. sapiens) | U2OS | ATCC | HTB-96 | |
| Cell line (H. sapiens) | K562 | ATCC | CCL-243 | |
| Genetic reagent (H. sapiens) | HEK-BEAR-GFP | Tálas et al., 2021 | ||
| Genetic reagent (H. sapiens) | HEK-BEAR-mScarlet | Tálas et al., 2021 | ||
| Cell line (H. sapiens) | HUES9 | Dr. Douglas Melton | ||
| Chemical compound, drug | Turbofect | Thermo Fischer Scientific Inc | R0531 | |
| Commercial assay or kit | Qubit dsDNA HS Assay Kit | Thermo Fischer Scientific Inc | Q32854 | |
| Commercial assay or kit | SF and SE Cell Line 4D-Nucleofector X Kit S | Lonza | V4XC-2032V4XC-1032 | |
| Recombinant DNA reagent | Plasmids | This study | see Supplementary file 2 | |
| Sequence-based reagents | DNA oligonucleotides | This study | see Supplementary file 3 | |
| Chemical compound, drug | Q5 High-Fidelity DNA Polymerase | New England Biolabs Inc | M0491L | |
| Chemical compound, drug | HiFi Assembly Master Mix | New England Biolabs Inc | E2621X | |
| Chemical compound, drug | KAPA Universal qPCR Master Mix | KAPA Biosystems | KK4602 | |
| Strain, strain background (Escherichia coli) | NEB5-alpha competent cells | New England Biolabs Inc | C2987I | |
| Recombinant DNA reagent | pCMV-PE2 | Addgene | (#132775) | |
| Recombinant DNA reagent | pAT9624-BEAR-cloning | Addgene | (#162986) | |
| Recombinant DNA reagent | pAT9651-BEAR-GFP | Addgene | (#162989) | |
| Recombinant DNA reagent | pAT9752-BEAR-mScarlet | Addgene | (#162991) | |
| Recombinant DNA reagent | pAT9658-sgRNA-mCherry | Addgene | (#162987) | |
| Recombinant DNA reagent | pAT9679-sgRNA-BFP | Addgene | (#162988) | |
| Recombinant DNA reagent | pX330-Flag-wtSpCas9-H840A | Addgene | (#80453) | |
| Recombinant DNA reagent | pX330-Flag-dSpCas9 | Addgene | (#92113) | |
| Recombinant DNA reagent | pX330-Flag-wtSpCas9 | Addgene | (#92353) |
Additional files
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Supplementary file 1
Primers and PCR condition to detect PEAR plasmid integration.
Forward and reverse PCR primers, PCR product sizes, and a detailed PCR protocol suitable for detecting integrated PEAR plasmids are provided.
- https://cdn.elifesciences.org/articles/69504/elife-69504-supp1-v1.docx
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Supplementary file 2
List of plasmids used in this study.
This file contains all of the plasmids used in the study with references and additional comments.
- https://cdn.elifesciences.org/articles/69504/elife-69504-supp2-v1.docx
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Supplementary file 3
List of oligonucleotides used in this study.
This file contains the oligonucleotide sequences used for molecular cloning or PCR.
- https://cdn.elifesciences.org/articles/69504/elife-69504-supp3-v1.docx
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Supplementary file 4
Sample indexing for NGS.
This file contains the necessary information to identify the NGS samples used in this study.
- https://cdn.elifesciences.org/articles/69504/elife-69504-supp4-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69504/elife-69504-transrepform1-v1.docx
