atp1a1 q118r  (Addgene inc)

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic for the co-enrichment of CRISPR-driven editing events at a GOI. b Schematic representation of the ATP1A1 locus regions targeted by SpCas9. The first and third extracellular loops of the Na + /K + ATPase are encoded by ATP1A1 exon 4 and 17, respectively. The relative levels of resistance to ouabain conferred by different ATP1A1 mutations in K562 cells are shown. c PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were transfected with PE3 vectors targeting ATP1A1 exon 17 (T804N) and the indicated GOI. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 0.5 µM ouabain until all non-resistant cells were eliminated. d Same as in c but co-targeting was performed via ATP1A1 exon 4 (Q118R). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: CRISPR, Sequencing, Transfection, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of the strategy for performing successive rounds of co-selection. Cells harboring modifications at ATP1A1 (T804N) and GOI A are first co-selected with 0.5 µM ouabain. Following the first round, a subsequent round of co-selection occurs at GOI B via modification at ATP1A1 (Q118R) with 100 µM ouabain. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells harboring the ATP1A1 -T804N and RUNX1 + 1 ATG insertion (GOI A) modifications (Fig. ) were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and the indicated GOI B. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. c Same as in b with K562 cells harboring the ATP1A1 -T804N and EMX1 + 1 G to C (GOI A) modifications (See Fig. ). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: Selection, Modification, Sequencing, Transfection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of mScarlet-I mTOR Signaling Indicator (mSc-TOSI) reporter degradation under mTORC1 signaling. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. n = 2 independent biological replicates performed at different times. c Histogram plot of mSc-TOSI intensity in homozygous single cell-derived K562 clones harboring MTOR hyperactivating mutations. d Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation. e Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -E2419K hyperactivating mutation. f Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -I2017T hyperactivating mutation. Where indicated, cells were treated for 24 h with 50 nM rapamycin or 50 nM AZD8055 before FACS analysis. Representative images are from one of two independent biological replicates performed at different times with equivalent results (see Supplementary Figs. and ). Source data are provided in the Source Data file.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: Sequencing, Stable Transfection, Expressing, Transfection, Selection, Derivative Assay, Clone Assay, Mutagenesis

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the large insertions observed at MTOR exon 44 from single cell-derived K562 clones harboring the MTOR -I2017T hyperactivating mutation. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. TOPO cloning and Sanger sequencing were performed to characterize the large insertions. The pegRNA and its PAM sequence are represented in orange and dark orange, respectively. The nick sgRNA and its PAM sequence are represented in blue and dark blue, respectively. b Same as in a for large insertions at MTOR exon 53 from single cell-derived K562 clones harboring the MTOR -E2419K hyperactivating mutation.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: Derivative Assay, Clone Assay, Mutagenesis, Stable Transfection, Expressing, Transfection, Isolation, Selection, Sequencing

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the complementary 5′ and 3′ single-stranded DNA overhangs generated with the PE3-T804N strategy at ATP1A1 exon 17 (PAM-In configuration) and the PE3-Q118R strategy at ATP1A1 exon 4 (PAM-Out configuration), respectively. b PCR-based genotyping of ATP1A1 exon 17 and 4 from single cell-derived MTOR -F2108L/I2017T K562 clones. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. n = 16 single cell-derived clones from one experiment. c Same as in b with single cell-derived MTOR -F2108L/E2419K K562 clones. n = 16 single cell-derived clones from one experiment. d Schematic representation of pegRNA and nick sgRNA target sites with PAMs facing inwards (PAM-In configuration) and outwards (PAM-Out configuration) at ATP1A1 exon 4. e PCR-based genotyping of ATP1A1 exon 4 from single cell-derived K562 clones targeted with PAM-out and PAM-in configurations using different nick sgRNAs. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 0.5 µM ouabain and genomic DNA was harvested after co-selection. n = 17 single cell-derived clones for each condition from one experiment. Ins, insertion. Del, deletion.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: Generated, Derivative Assay, Clone Assay, Isolation, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the EBFP to EGFP reporter system using different nick sgRNAs with PAMs facing outwards (PAM-Out) or inwards (PAM-In) towards the pegRNA. b Schematic of the FACS-based quantification of EBFP to EGFP conversion via PE. In K562 cells homozygous for EBFP integration at ATP1A1 (triallelic), FACS-based analysis reports PE outcomes indirectly; (i) EBFP(+)/EGFP(+) cells result from monoallelic or biallelic PE, (ii) EBFP(−)/EGFP(+) cells are generated by either triallelic PE or combinations of mono- and biallelic PE along with indels*, (iii) EBFP(−)/EGFP(−) occur from indels on the three copies of the reporter. Indels are defined broadly in this context as any edits that inactivates the reporter. c FACS-based quantification of EBFP to EGFP conversion via PE after co-selection. K562 cells stably expressing the EBFP reporter from the ATP1A1 locus were transfected with PE3max vectors targeting ATP1A1 exon 4 (pegRNA-Q118R_v1) and EBFP (epegRNA). Cells were treated with 100 µM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. n = 3 independent biological replicates performed at different times with equivalent results (see Supplementary Figs. ). d PCR-based genotyping of EGFP(+) single cell-derived K562 clones targeted with the PE2 or PE3b strategy. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA from EGFP(+) clones was harvested after co-selection. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. n = 17 single cell-derived clones for each condition from one experiment. e PCR-based genotyping of EBFP from single K562 cell-derived clones after sorting for EBFP(−)/EGFP(+) cells. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. Larger insertions and deletions are indicated, and homozygous single cell-derived clones are highlighted in bold and green. PE alleles harboring pegRNA scaffold incorporation are indicated (si). n = 17 and 16 single cell-derived clones from one experiment for the PE3-G3 and PE3-G6 conditions, respectively. Ins, insertion. Del, deletion.

Article Snippet: U6-pegRNA cassette expressing the ATP1A1 -Q118R and T804N cassettes were cloned upstream of the U6-RFP-acceptor cassette to create ATP1A1 _G4_Q118R_Dual_pegRNA (Addgene 173199) and ATP1A1 _G6_T804N _Dual_pegRNA (Addgene 173200).

Techniques: Generated, Selection, Stable Transfection, Expressing, Transfection, Derivative Assay, Clone Assay, Isolation, Sequencing

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic for the co-enrichment of CRISPR-driven editing events at a GOI. b Schematic representation of the ATP1A1 locus regions targeted by SpCas9. The first and third extracellular loops of the Na + /K + ATPase are encoded by ATP1A1 exon 4 and 17, respectively. The relative levels of resistance to ouabain conferred by different ATP1A1 mutations in K562 cells are shown. c PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were transfected with PE3 vectors targeting ATP1A1 exon 17 (T804N) and the indicated GOI. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 0.5 µM ouabain until all non-resistant cells were eliminated. d Same as in c but co-targeting was performed via ATP1A1 exon 4 (Q118R). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: CRISPR, Sequencing, Transfection, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of the strategy for performing successive rounds of co-selection. Cells harboring modifications at ATP1A1 (T804N) and GOI A are first co-selected with 0.5 µM ouabain. Following the first round, a subsequent round of co-selection occurs at GOI B via modification at ATP1A1 (Q118R) with 100 µM ouabain. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells harboring the ATP1A1 -T804N and RUNX1 + 1 ATG insertion (GOI A) modifications (Fig. ) were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and the indicated GOI B. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. c Same as in b with K562 cells harboring the ATP1A1 -T804N and EMX1 + 1 G to C (GOI A) modifications (See Fig. ). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Selection, Modification, Sequencing, Transfection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of mScarlet-I mTOR Signaling Indicator (mSc-TOSI) reporter degradation under mTORC1 signaling. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. n = 2 independent biological replicates performed at different times. c Histogram plot of mSc-TOSI intensity in homozygous single cell-derived K562 clones harboring MTOR hyperactivating mutations. d Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation. e Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -E2419K hyperactivating mutation. f Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -I2017T hyperactivating mutation. Where indicated, cells were treated for 24 h with 50 nM rapamycin or 50 nM AZD8055 before FACS analysis. Representative images are from one of two independent biological replicates performed at different times with equivalent results (see Supplementary Figs. and ). Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Sequencing, Stable Transfection, Expressing, Transfection, Selection, Derivative Assay, Clone Assay, Mutagenesis

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the large insertions observed at MTOR exon 44 from single cell-derived K562 clones harboring the MTOR -I2017T hyperactivating mutation. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. TOPO cloning and Sanger sequencing were performed to characterize the large insertions. The pegRNA and its PAM sequence are represented in orange and dark orange, respectively. The nick sgRNA and its PAM sequence are represented in blue and dark blue, respectively. b Same as in a for large insertions at MTOR exon 53 from single cell-derived K562 clones harboring the MTOR -E2419K hyperactivating mutation.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Derivative Assay, Clone Assay, Mutagenesis, Stable Transfection, Expressing, Transfection, Isolation, Selection, Sequencing

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the complementary 5′ and 3′ single-stranded DNA overhangs generated with the PE3-T804N strategy at ATP1A1 exon 17 (PAM-In configuration) and the PE3-Q118R strategy at ATP1A1 exon 4 (PAM-Out configuration), respectively. b PCR-based genotyping of ATP1A1 exon 17 and 4 from single cell-derived MTOR -F2108L/I2017T K562 clones. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. n = 16 single cell-derived clones from one experiment. c Same as in b with single cell-derived MTOR -F2108L/E2419K K562 clones. n = 16 single cell-derived clones from one experiment. d Schematic representation of pegRNA and nick sgRNA target sites with PAMs facing inwards (PAM-In configuration) and outwards (PAM-Out configuration) at ATP1A1 exon 4. e PCR-based genotyping of ATP1A1 exon 4 from single cell-derived K562 clones targeted with PAM-out and PAM-in configurations using different nick sgRNAs. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 0.5 µM ouabain and genomic DNA was harvested after co-selection. n = 17 single cell-derived clones for each condition from one experiment. Ins, insertion. Del, deletion.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Generated, Derivative Assay, Clone Assay, Isolation, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the EBFP to EGFP reporter system using different nick sgRNAs with PAMs facing outwards (PAM-Out) or inwards (PAM-In) towards the pegRNA. b Schematic of the FACS-based quantification of EBFP to EGFP conversion via PE. In K562 cells homozygous for EBFP integration at ATP1A1 (triallelic), FACS-based analysis reports PE outcomes indirectly; (i) EBFP(+)/EGFP(+) cells result from monoallelic or biallelic PE, (ii) EBFP(−)/EGFP(+) cells are generated by either triallelic PE or combinations of mono- and biallelic PE along with indels*, (iii) EBFP(−)/EGFP(−) occur from indels on the three copies of the reporter. Indels are defined broadly in this context as any edits that inactivates the reporter. c FACS-based quantification of EBFP to EGFP conversion via PE after co-selection. K562 cells stably expressing the EBFP reporter from the ATP1A1 locus were transfected with PE3max vectors targeting ATP1A1 exon 4 (pegRNA-Q118R_v1) and EBFP (epegRNA). Cells were treated with 100 µM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. n = 3 independent biological replicates performed at different times with equivalent results (see Supplementary Figs. ). d PCR-based genotyping of EGFP(+) single cell-derived K562 clones targeted with the PE2 or PE3b strategy. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA from EGFP(+) clones was harvested after co-selection. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. n = 17 single cell-derived clones for each condition from one experiment. e PCR-based genotyping of EBFP from single K562 cell-derived clones after sorting for EBFP(−)/EGFP(+) cells. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. Larger insertions and deletions are indicated, and homozygous single cell-derived clones are highlighted in bold and green. PE alleles harboring pegRNA scaffold incorporation are indicated (si). n = 17 and 16 single cell-derived clones from one experiment for the PE3-G3 and PE3-G6 conditions, respectively. Ins, insertion. Del, deletion.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Generated, Selection, Stable Transfection, Expressing, Transfection, Derivative Assay, Clone Assay, Isolation, Sequencing

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic for the co-enrichment of CRISPR-driven editing events at a GOI. b Schematic representation of the ATP1A1 locus regions targeted by SpCas9. The first and third extracellular loops of the Na + /K + ATPase are encoded by ATP1A1 exon 4 and 17, respectively. The relative levels of resistance to ouabain conferred by different ATP1A1 mutations in K562 cells are shown. c PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were transfected with PE3 vectors targeting ATP1A1 exon 17 (T804N) and the indicated GOI. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 0.5 µM ouabain until all non-resistant cells were eliminated. d Same as in c but co-targeting was performed via ATP1A1 exon 4 (Q118R). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: CRISPR, Sequencing, Transfection, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of the strategy for performing successive rounds of co-selection. Cells harboring modifications at ATP1A1 (T804N) and GOI A are first co-selected with 0.5 µM ouabain. Following the first round, a subsequent round of co-selection occurs at GOI B via modification at ATP1A1 (Q118R) with 100 µM ouabain. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells harboring the ATP1A1 -T804N and RUNX1 + 1 ATG insertion (GOI A) modifications (Fig. ) were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and the indicated GOI B. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. c Same as in b with K562 cells harboring the ATP1A1 -T804N and EMX1 + 1 G to C (GOI A) modifications (See Fig. ). n = 2 independent biological replicates performed at different times. Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Selection, Modification, Sequencing, Transfection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic of mScarlet-I mTOR Signaling Indicator (mSc-TOSI) reporter degradation under mTORC1 signaling. b PE and small indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 µM ouabain until all non-resistant cells were eliminated. n = 2 independent biological replicates performed at different times. c Histogram plot of mSc-TOSI intensity in homozygous single cell-derived K562 clones harboring MTOR hyperactivating mutations. d Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation. e Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -E2419K hyperactivating mutation. f Same as in c with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation and the MTOR -I2017T hyperactivating mutation. Where indicated, cells were treated for 24 h with 50 nM rapamycin or 50 nM AZD8055 before FACS analysis. Representative images are from one of two independent biological replicates performed at different times with equivalent results (see Supplementary Figs. and ). Source data are provided in the Source Data file.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Sequencing, Stable Transfection, Expressing, Transfection, Selection, Derivative Assay, Clone Assay, Mutagenesis

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the large insertions observed at MTOR exon 44 from single cell-derived K562 clones harboring the MTOR -I2017T hyperactivating mutation. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. TOPO cloning and Sanger sequencing were performed to characterize the large insertions. The pegRNA and its PAM sequence are represented in orange and dark orange, respectively. The nick sgRNA and its PAM sequence are represented in blue and dark blue, respectively. b Same as in a for large insertions at MTOR exon 53 from single cell-derived K562 clones harboring the MTOR -E2419K hyperactivating mutation.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Derivative Assay, Clone Assay, Mutagenesis, Stable Transfection, Expressing, Transfection, Isolation, Selection, Sequencing

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the complementary 5′ and 3′ single-stranded DNA overhangs generated with the PE3-T804N strategy at ATP1A1 exon 17 (PAM-In configuration) and the PE3-Q118R strategy at ATP1A1 exon 4 (PAM-Out configuration), respectively. b PCR-based genotyping of ATP1A1 exon 17 and 4 from single cell-derived MTOR -F2108L/I2017T K562 clones. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA was harvested after co-selection. n = 16 single cell-derived clones from one experiment. c Same as in b with single cell-derived MTOR -F2108L/E2419K K562 clones. n = 16 single cell-derived clones from one experiment. d Schematic representation of pegRNA and nick sgRNA target sites with PAMs facing inwards (PAM-In configuration) and outwards (PAM-Out configuration) at ATP1A1 exon 4. e PCR-based genotyping of ATP1A1 exon 4 from single cell-derived K562 clones targeted with PAM-out and PAM-in configurations using different nick sgRNAs. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 0.5 µM ouabain and genomic DNA was harvested after co-selection. n = 17 single cell-derived clones for each condition from one experiment. Ins, insertion. Del, deletion.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Generated, Derivative Assay, Clone Assay, Isolation, Selection

Journal: Nature Communications

Article Title: Marker-free co-selection for successive rounds of prime editing in human cells

doi: 10.1038/s41467-022-33669-z

Figure Lengend Snippet: a Schematic representation of the EBFP to EGFP reporter system using different nick sgRNAs with PAMs facing outwards (PAM-Out) or inwards (PAM-In) towards the pegRNA. b Schematic of the FACS-based quantification of EBFP to EGFP conversion via PE. In K562 cells homozygous for EBFP integration at ATP1A1 (triallelic), FACS-based analysis reports PE outcomes indirectly; (i) EBFP(+)/EGFP(+) cells result from monoallelic or biallelic PE, (ii) EBFP(−)/EGFP(+) cells are generated by either triallelic PE or combinations of mono- and biallelic PE along with indels*, (iii) EBFP(−)/EGFP(−) occur from indels on the three copies of the reporter. Indels are defined broadly in this context as any edits that inactivates the reporter. c FACS-based quantification of EBFP to EGFP conversion via PE after co-selection. K562 cells stably expressing the EBFP reporter from the ATP1A1 locus were transfected with PE3max vectors targeting ATP1A1 exon 4 (pegRNA-Q118R_v1) and EBFP (epegRNA). Cells were treated with 100 µM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. n = 3 independent biological replicates performed at different times with equivalent results (see Supplementary Figs. ). d PCR-based genotyping of EGFP(+) single cell-derived K562 clones targeted with the PE2 or PE3b strategy. Single cell-derived clones were isolated in methylcellulose-based semi-solid RPMI media supplemented with 100 µM ouabain and genomic DNA from EGFP(+) clones was harvested after co-selection. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. n = 17 single cell-derived clones for each condition from one experiment. e PCR-based genotyping of EBFP from single K562 cell-derived clones after sorting for EBFP(−)/EGFP(+) cells. The number of precise prime edited alleles was determined from BEAT Sanger sequencing trace analysis and small indels were analysed with DECODR. Larger insertions and deletions are indicated, and homozygous single cell-derived clones are highlighted in bold and green. PE alleles harboring pegRNA scaffold incorporation are indicated (si). n = 17 and 16 single cell-derived clones from one experiment for the PE3-G3 and PE3-G6 conditions, respectively. Ins, insertion. Del, deletion.

Article Snippet: The U6- ATP1A1 nick sgRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Generated, Selection, Stable Transfection, Expressing, Transfection, Derivative Assay, Clone Assay, Isolation, Sequencing

Journal: bioRxiv

Article Title: Marker-free coselection for successive rounds of prime editing in human cells

doi: 10.1101/2021.11.02.464583

Figure Lengend Snippet: ( a ) Schematic for the co-enrichment of CRISPR-driven editing events at a GOI. ( b ) Schematic representation of the ATP1A1 locus regions targeted by SpCas9. The first and third extracellular loops of the Na + /K + ATPase are encoded by ATP1A1 exon 4 and 17, respectively. The relative levels of resistance to ouabain conferred by different ATP1A1 mutations in K562 cells are shown. ( c ) FACS-based quantification of EBFP to EGFP conversion. K562 cells stably expressing EBFP were transfected with nuclease vectors and ssODN donors targeting ATP1A1 and EBFP. Where indicated, cells were treated with 0.5 μM ouabain starting 3 days post-transfection and expanded until all non-resistant cells were eliminated. ( d ) FACS-based quantification of HDR-driven tagging of NPM1 with mNeonGreen. K562 cells were transfected with nuclease vectors and donors targeting ATP1A1 and NPM1 . Where indicated, cells were treated with 0.5 μM ouabain starting 3 days post-transfection and expanded until all non-resistant cells were eliminated. ( e ) Same as in ( d ) for HDR-driven tagging of LMNA with mScarlet-I. PGK1 , human phosphoglycerate kinase 1 promoter. PA, polyadenylation signal. HA, homology arm. E, exon.

Article Snippet: The U6- ATP1A1 nick gRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: CRISPR, Stable Transfection, Expressing, Transfection

Journal: bioRxiv

Article Title: Marker-free coselection for successive rounds of prime editing in human cells

doi: 10.1101/2021.11.02.464583

Figure Lengend Snippet: ( a ) Schematic representation of the targeted integration of mScarlet-I mTOR Signaling Indicator (mSc-TOSI) in antisense orientation within intron 17 of ATP1A1 . The T804N mutation is introduced via the left homology arm (HA) and transgene expression is driven by the PGK1 promoter. ( b ) FACS-based quantification of mSc-TOSI knock-in at ATP1A1 intron 17. K562 cells were transfected with SpCas9 and the donor and treated with or without 0.5 μM ouabain starting 3 days post-transfection until all non-resistant cells were eliminated. ( c ) Schematic representation of mSc-TOSI targeting to the AAVS1 locus. ( d ) FACS-based quantification of mSc-TOSI knock-in at AAVS1 . K562 cells were transfected with SpCas9 and a donor targeting AAVS1 and treated with or without 0.5 μg/ml puromycin starting 3 days post-transfection until all non-resistant cells were eliminated. ( e ) Schematic of mSc-TOSI degradation under mTORC1 signaling. ( f ) mSc-TOSI fluorescence intensity after stable integration at the ATP1A1 and AAVS1 loci in presence and absence of rapamycin. Where indicated, cells were treated with 500 nM rapamycin or vehicle control for 24 hours before FACS analysis. PGK1 , human phosphoglycerate kinase 1 promoter. PA, polyadenylation signal. HA, homology arm. E, exon.

Article Snippet: The U6- ATP1A1 nick gRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Mutagenesis, Expressing, Knock-In, Transfection, Fluorescence

Journal: bioRxiv

Article Title: Marker-free coselection for successive rounds of prime editing in human cells

doi: 10.1101/2021.11.02.464583

Figure Lengend Snippet: ( a ) Schematic of the dual and tandem U6-driven pegRNAs and sgRNAs expression vectors along with the PE2 expression vector used to target ATP1A1 and a GOI. ( b ) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were transfected with PE3 vectors targeting ATP1A1 exon 17 (T804N) and the indicated GOI. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 0.5 μM ouabain until all non-resistant cells were eliminated. ( c ) Same as in ( b ) but co-targeting was performed via ATP1A1 exon 4 (Q118R). These experiments were performed twice and at different times with equivalent results (see also Supplementary Fig. 8 ).

Article Snippet: The U6- ATP1A1 nick gRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Expressing, Plasmid Preparation, Sequencing, Transfection, Selection

Journal: bioRxiv

Article Title: Marker-free coselection for successive rounds of prime editing in human cells

doi: 10.1101/2021.11.02.464583

Figure Lengend Snippet: ( a ) Schematic of the strategy for performing successive rounds of coselection. Cells harboring modifications at ATP1A1 (T804N) and GOI A are first coselected with 0.5 μM ouabain. Following the first round, a subsequent round of coselection occurs at GOI B via modification at ATP1A1 (Q118R) with 100 μM ouabain. ( b ) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells harboring the ATP1A1 -T804N and RUNX1 +1 ATG insertion (GOI A) modifications were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and the indicated GOI B. Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 μM ouabain until all non-resistant cells were eliminated. ( c ) Same as in ( b ) with K562 cells harboring the ATP1A1 -T804N and EMX1 +1 G to C (GOI A) modifications. These experiments were performed twice and at different times with equivalent results (see also Supplementary Fig. 10 ).

Article Snippet: The U6- ATP1A1 nick gRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Modification, Sequencing, Transfection, Selection

Journal: bioRxiv

Article Title: Marker-free coselection for successive rounds of prime editing in human cells

doi: 10.1101/2021.11.02.464583

Figure Lengend Snippet: ( a ) Schematic representation of reporter cell line engineering. K562 cells stably expressing the mSc-TOSI reporter from ATP1A1 intron 17 are first selected with 0.5 μM ouabain. Then, coselection for PE to insert the indicated mutations at MTOR is performed with 100 μM ouabain. ( b ) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells stably expressing the mSc-TOSI reporter were transfected with PE3 vectors targeting ATP1A1 exon 4 (Q118R) and MTOR . Genomic DNA was harvested 3 days post-transfection (before selection) and cells were treated (ouabain) or not (untreated) with 100 μM ouabain until all non-resistant cells were eliminated. This experiment was performed twice and at different times with equivalent results (see also Supplementary Fig. 11 ). ( c ) Histogram plot of mSc-TOSI intensity in homozygous single cell-derived K562 clones harboring MTOR hyperactivating mutations. ( d ) Same as in ( c ) with a homozygous clone harboring the MTOR -F2108L rapamycin resistance mutation. Where indicated, cells were treated for 24 hours with 50 nM rapamycin or 50 nM AZD8055 before FACS analysis.

Article Snippet: The U6- ATP1A1 nick gRNA cassettes were cloned upstream of the U6-BbsI-sgRNA cassette to create ATP1A1 _G3_Dual_sgRNA (Addgene 173202) and ATP1A1 _G8 _Dual_sgRNA (Addgene 178104).

Techniques: Stable Transfection, Expressing, Sequencing, Transfection, Selection, Derivative Assay, Clone Assay, Mutagenesis

Journal: Molecular cell

Article Title: CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

doi: 10.1016/j.molcel.2017.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: B270 (containing sgRNA targeting ATP1A1 + empty sgRNA-expressing cassette) , This paper , Available in Addgene.

Techniques: Virus, Subcloning, Recombinant, Transfection, DNA Extraction, PCR Cloning, Cloning, Plasmid Preparation, Software