Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Schematic of prime editing. b , Schematic of the FACS reporter of prime editing. c , Gene-level phenotypes from genome-scale CRISPRi screen performed in FACS reporter cells with the SaPE2 editor, +7 GG-to-CA edit and the PE3 approach. Phenotypes represent enrichment of normalized sgRNA counts in GFP + over GFP – populations after prime editing (average for the top three sgRNAs per gene). Hit genes (FDR ≤ 0.01) were identified using CRISPhieRmix . Pseudogene controls generated from randomly selected non-targeting (NT) sgRNAs. d , Quantification of CRISPRi-mediated La depletion. Reverse transcription followed by quantitative PCR (RT–qPCR) of RNA from K562 CRISPRi cells with integrated MCS reporter. Data are normalized to ACTB and are presented relative to a non-targeting sgRNA (NT1). La1 and La2, La -targeting sgRNAs. e , Percentages of prime editing outcomes produced at the integrated MCS reporter using the SaPE2 editor with or without depletion of La in K562 CRISPRi cells. Percentages of intended prime editing without indels (left), indels with the intended prime edit (middle) and indels without the intended edit (right) plotted separately. Editing components delivered by plasmid transfection in c and e . Horizontal bars in d indicate geometric means ( n = 3 independent biological replicates). Data and error bars in e indicate mean ± s.d. ( n = 3 independent biological replicates). Image of the prime editor protein in a adapted from ref. , Elsevier, under a Creative Commons licence CC BY 4.0 . Images of DNA and pegRNA in a adapted from ref. , Elsevier.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Produced, Plasmid Preparation, Transfection

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , b , Percentages of prime editing outcomes produced at integrated FACS reporter with pegRNA (left) or epegRNA (right, tevopreQ 1 ) in K562 CRISPRi cells after transduction of the indicated sgRNA. Intended editing quantified by flow cytometry (a) or sequencing (b). c , Schematic of workflow used to engineer K562 clonal cell lines with PEmax expressed constitutively from the AAVS1 safe-harbor locus (parental K562 PEmax cells). d , Western blot analysis of K562 cells constitutively expressing PEmax (K562 PEmax parental) and clones with genetic disruption of La (La-ko1-La-ko5). Asterisks, cell lines used in this study. Images are from the same blot as presented in Fig. . For additional details on imaging, see and Supplementary Fig. . e , Sequences and frequencies of alleles observed at the La locus in the La-knockout clones used in this study (La-ko3 through La-ko5). Analysis performed with CRISPResso2 . f , Cumulative population doublings of parental, La-ko4, and La-ko5 K562 PEmax cells. g , Flow cytometry analysis of GFP expressed from the PEmax construct at the AAVS1 locus in K562 PEmax parental, La-ko3, La-ko4, and La-ko5 cells. Data collected from cells prior to transfection for experiment depicted in Fig. . h , Percentages of prime editing (PE3) outcomes across ten edits with pegRNAs (top) or epegRNAs (bottom) at five genomic loci in HEK293T cells with and without depletion of La by siRNA. Fold-changes in outcome frequencies presented in Fig. . Editing components delivered by plasmid transfection in a, b and h. Data and error bars in a, b and h indicate mean ± s.d. (n = 4 independent biological replicates). Data in d, e and g depict results from characterizations of n = 1 cell lines. Percentages in f indicate relative mean ± s.d. (n = 3 independent biological replicates measured across an 8-day time course) of daily fold changes in cell numbers, essentially the relative percentages of cells to expect after one day of growth for La-ko4 and La-ko5 compared with parental K562 PEmax cells. P -values in h are from one-tailed unpaired Student’s t -test.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Produced, Transduction, Flow Cytometry, Sequencing, Western Blot, Expressing, Clone Assay, Disruption, Imaging, Knock-Out, Construct, Transfection, Plasmid Preparation, One-tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Western blot analysis of K562 cells constitutively expressing PEmax (K562 PEmax parental) and clones with genetic disruption of La (La-ko1 through La-ko5). Asterisks indicate cell lines used in this study. See also Extended Data Fig. . b , Percentages of prime editing outcomes at indicated genomic loci. pegRNAs and epegRNAs (evopreQ 1 ) were delivered as plasmids without or with MLH1dn (PE2 or PE4, respectively). c , Percentages of prime editing outcomes with or without ectopic expression of La . Expression plasmids for La or mRFP control were delivered alongside plasmids encoding pegRNA or epegRNA (evopreQ 1 ). The PE2 approach was used. d , Quantification of RNAi-mediated La depletion. RT–qPCR from HEK293T cells. Data normalized to ACTB and presented relative to the non-targeting (NT) siRNA pool. e , Fold changes in prime editing outcomes across ten PE3 edits (substitutions, insertions and deletions) at five genomic loci in HEK293T cells with or without La depletion. Editing percentages are presented in Extended Data Fig. . f , Top, schematic of the MCS reporter, including distances between the predicted SaCas9 cut site and sequences required for GFP expression. Bottom, flow cytometry analysis of MCS reporter cells with and without CRISPRi-mediated La depletion after induction of SaCas9-mediated DSB and unedited controls. Quantification presented in Extended Data Fig. . g , h , Fold changes in editing outcomes induced with pegRNA ( g ) or sgRNA ( h ) using SaABE8e, SaBE4, SaCas9 or SaPE2 (PE4 approach, g only) in La-ko4 relative to parental K562 PEmax cells (intended edits only). Editing percentages presented in Extended Data Fig. . Editing components were delivered by plasmid transfection in b , c and e – h . Data and error bars in b and c indicate the mean ± s.d. ( n = 4 and 3 independent biological replicates, respectively). Horizontal bars in d and e indicate geometric means ( n = 3 independent biological replicates) and medians of fold changes (10 edits, each with n = 4 independent biological replicates plotted individually), respectively. Data in g and h represent ratios of means for individual editing outcomes ( n = 3 independent biological replicates for each outcome).

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Clone Assay, Disruption, Quantitative RT-PCR, Flow Cytometry, Plasmid Preparation, Transfection

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Percentages of GFP- cells within indicated cell populations arising from SaCas9-induced DSBs at a stably integrated MCS reporter in K562 CRISPRi cells. CRISPRi sgRNAs were delivered by lentiviral transduction. Editing components (SaCas9, +7 GG to CA pegRNA) were delivered by plasmid transfection. Representative flow cytometry data from each condition and unedited controls also presented in Fig. . b , Quantification of SaCas9-induced indels at stably integrated MCS reporter described in a. c-f , Percentages of intended editing achieved in K562 PEmax parental and La - ko4 cells using SaPE2 with the PE4 approach, SaCas9, SaBE4, and SaABE8e across four genomic loci, HEK3 (c), EMX1 (d), FANCF (e) and HBB (f). The same pegRNA or sgRNA expression plasmid was used for all editing systems at each target, with select combinations excluded (SaPE2 with PE4 approach with any sgRNA and SaBE4 at EMX1). Relative editing for each intended outcome presented in Fig. and . Data and error bars in a-f indicate mean ± s.d. (n = 3 independent biological replicates). P -values in a and b are from two-tailed unpaired Student’s t -test.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Transfection, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Domain architectures of La and mutants. NRE, nuclear retention element Linker, SGGS. b , Percentages of prime editing outcomes with or without ectopic expression of La or mutants depicted in a . Expression plasmids were delivered to indicated cells alongside the plasmid encoding the DNMT1 +5 G-to-T pegRNA. c , Schematic of RNA with chemical modifications (bold); specifically, phosphorothioate bonds (asterisks in sequence representation) and 2′-OMe modifications (‘m’ in sequence representation). d , Percentages of prime editing outcomes produced using 100 pmole of synthetic pegRNAs with indicated 3′ end configurations. e , Fold changes in average intended prime editing at four genomic loci in La-ko4 cells relative to parental K562 PEmax cells produced using 100 pmole of synthetic pegRNA with the indicated 3′ end configurations. Editing percentages provided in Extended Data Fig. . f , Model of La interaction with pegRNAs. The PE2 approach was used in b , d , e . Underlining in d , e indicates particular 3′ end configuration patterns. Data and error bars in b and d indicate the mean ± s.d. ( n = 2–3 independent biological replicates). Vertical bars in e indicate medians (4 edits) of ratios of means ( n = 3 independent biological replicates for each edit). P values in d are from one-tailed unpaired Student’s t -test. Image of pegRNA in f adapted from ref. , Elsevier.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Expressing, Plasmid Preparation, Sequencing, Produced, One-tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Chemical structures of ribonucleotides linked by a phosphorothioate bond (left) or with substitution of ribose 2′-OH for 2′-O-methyl groups (2′-OCH 3 ) (right). b , Percentages of prime editing outcomes at the endogenous DNMT1 locus in parental K562 PEmax cells using one synthetic pegRNA with the indicated 3′ end configuration. Input was titrated from 0 to 500 pmole. c , d , Percentages of prime editing outcomes at the endogenous HEK3 (c) and DNMT1 (d) loci in K562 PEmax cells using 100 pmole of synthetic pegRNAs and 50 pmole of synthetic sgRNA (c only) with specified 3′ end sequences and chemical modifications. e , Percentages of prime editing outcomes at endogenous DNMT1, CXCR4, VEGFA, and RUNX1 loci in K562 PEmax parental and La-ko4 cells using 100 pmole of synthetic pegRNAs with indicated 3′ end configurations. Fold-changes in outcome frequencies also presented in Fig. . Data and error bars in b-e indicate mean ± s.d. (n = 3 independent biological replicates). P -values in c-e are from one-tailed unpaired Student’s t -test.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: One-tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data are from samples collected one and two days after transfection of eleven (e)pegRNAs in two sets. b , Fold changes in normalized counts of indicated biotypes in La-ko4 cells relative to parental K562 PEmax cells, from samples collected one and two days after transfection of eleven (e)pegRNAs in two sets. Counts were calculated per replicate for each set of (e)pegRNAs as the sums of properly aligned fragments classified as each biotype and normalized by total RNA counts. c , Schematic of minimum sequence defining each class of (e)pegRNA fragments from small RNA-seq (orange , cis -active; purple, trans -active). Representative sequence used ( i.e ., RUNX1 + 5 G to T pegRNA). Edit-encoding nucleotide (white base) and cryptic terminators (green asterisks) indicated. d , Plot (MA) of small RNA-seq data displaying mean normalized expression versus log 2 -fold change in expression of human genes and (e)pegRNA bins from La-ko4 cells relative to parental K562 PEmax cells. Data are from samples collected one (top) and two (bottom) days after transfection of plasmids encoding seven pegRNAs and four epegRNAs. Alignment categories are indicated (gray, human small RNA; orange, cis -active; purple, trans -active; green, premature termination) and genes with adjusted p -values ≤ 0.05 are highlighted in light gray. e , Coverage plots of small RNA-seq fragments for the pegRNA (left) or epegRNA (right) specifying RUNX1 + 5 G to T from specified cell lines collected one day after (e)pegRNA plasmid transfection. Data are normalized by counts of fragments from total human small RNA (top) or those within the corresponding bins: cis -active, trans -active, inactive (bottom). Nucleotide position 0 denotes the 5′ end of the RNA, and positions of the edit-encoding nucleotide (vertical solid line) and the start of PBS (vertical dashed line) are indicated. Shaded areas represent sgRNA sequence and Pol III terminator (pegRNA) or sgRNA sequence, linker, evopreQ 1 , and Pol III terminator (epegRNA). f , Coverage plots of small RNA-seq fragments for pegRNAs specifying RNF2 + 1 C to A (left), VEGFA + 5 G to T (middle) or FANCF + 5 G to T (right) from specified cell lines collected one day after (e)pegRNA plasmid transfection. Data are normalized by counts of fragments from total human small RNA (top) or those within the corresponding bins: cis -active, trans -active, inactive (bottom). Nucleotide position 0 denotes the 5′ end of the RNA, and positions of the edit-encoding nucleotide (vertical solid line) and the start of PBS (vertical dashed line) are indicated. Shaded areas represent sgRNA sequence and Pol III terminator. Data in a indicate means (n = 3 independent biological replicates). Horizontal bars in b indicate medians (12 data points per biotype, each biotype has n = 3 independent biological replicates for each day and each set of (e)pegRNAs). Data in d were calculated from n = 6 (VEGFA + 5 G to T) and 3 (all others) independent biological replicates and adjusted P -values were calculated by DESeq2 using the two-tailed Wald test with Bonferroni-Holm correction. Coverages in e and f represent n = 6 (VEGFA + 5 G to T) and 3 (all others) independent biological replicates. Image of pegRNA in c adapted from ref. , Springer Nature America.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: RNA Sequencing Assay, Transfection, Sequencing, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Composition of small RNA-seq libraries from K562 PEmax parental or La-ko4 cells. Data from samples collected one and two days after transfection of plasmid encoding a pegRNA or an epegRNA specifying mouse DNMT1 + 6 G to C. b . Fold changes in normalized counts of indicated biotypes in La-ko4 cells relative to parental K562 PEmax cells, from samples collected one and two days after transfection of plasmid encoding a pegRNA or an epegRNA specifying mouse DNMT1 + 6 G to C. Counts were calculated per replicate for the pegRNA and the epegRNA as the sums of properly aligned fragments classified as each biotype and normalized by total RNA counts. c , d , Coverage plots of small RNA-seq fragments for the pegRNA (left) or the epegRNA (right) specifying mouse DNMT1 + 6 G to C edit from specified cell lines, which lack the (e)pegRNA target, collected one (c) and two (d) days after (e)pegRNA plasmid transfection. Data are normalized by counts of fragments from total human small RNA (top) or those within the corresponding bins: cis -active, trans -active, inactive (bottom). Nucleotide position 0 denotes the 5′ end of the RNA, and positions of the edit-encoding nucleotide (vertical solid line) and the start of PBS (vertical dashed line) are indicated. Shaded areas represent sgRNA sequence, and Pol III terminator for the pegRNA and tevopreQ 1 plus Pol III terminator for the epegRNA. e , Percentages of cis -active fragments with the edit-encoding nucleotide for the pegRNA (left) and the epegRNA (right) specifying mouse DNMT1 + 6 G to C edit in K562 PEmax parental or La-ko4 cells without the (e)pegRNA target. Associated coverage plots presented in c and d. f , Percentages of prime editing outcomes in K562 PEmax parental and La-ko4 cells transduced with the mouse DNMT1 target and transfected with either the pegRNA or epegRNA plasmid specifying mouse DNMT1 + 6 G to C. Data are from samples collected on indicated days. Data in a indicate means (n = 4 independent biological replicates). Horizontal bars in b indicate medians (16 data points per biotype, each biotype has n = 4 independent biological replicates for the pegRNA and epegRNA on each day). Coverages depicted in c and d represent n = 4 independent biological replicates. Data and error bars in e and f indicate mean ± s.d. (n = 4 and 3 independent biological replicates, respectively). P -values in e are from two-tailed unpaired Student’s t -test.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: RNA Sequencing Assay, Transfection, Plasmid Preparation, Sequencing, Transduction, Two Tailed Test

Journal: Nature

Article Title: Improving prime editing with an endogenous small RNA-binding protein

doi: 10.1038/s41586-024-07259-6

Figure Lengend Snippet: a , Percentages of prime editing outcomes at the endogenous HEK3 and PRNP loci in K562 cells using PEmax, PE7 or PE7 mutant. Editing components delivered by plasmid transfection. Cells from this experiment were also used for analyses in b-i. b , Percentages of viable K562 cells quantified by flow cytometry one, two, and three days after transfection of pegRNA plasmid specifying either HEK3 + 1 T to A or PRNP + 6 G to T and PEmax, PE7, or PE7 mutant encoding plasmid. c , Cumulative population doublings of K562 cells two and three days after transfection of pegRNA plasmid specifying either HEK3 + 1 T to A or PRNP + 6 G to T and PEmax, PE7, or PE7 mutant encoding plasmid. d-f , Plot (MA) of RNA-seq data displaying mean normalized gene expression versus log 2 -fold change in gene expression from K562 cells edited with PE7 relative to PEmax (d), PE7 relative to PE7 mutant (e), and PEmax relative to PE7 mutant (f). Analyses were performed with cells edited using two different pegRNAs, one specifying HEK3 + 1 T to A (top) and one specifying PRNP + 6 G to T (bottom). Upregulated and downregulated genes with adjusted P -values ≤ 0.05 are highlighted in red and blue, respectively. g-i , Venn diagrams of differentially expressed genes ( p ≤ 0.05) in K562 cells edited at two different loci across three comparisons: PE7 relative to PEmax (g), PE7 relative to PE7 mutant (h), and PEmax relative to PE7 mutant (i). Bolded genes represent those significantly changed in more than one of the indicated comparisons. Data and error bars in a indicate mean ± s.d. (n = 4 independent biological replicates). Horizontal bars in b and c indicate means (n = 4 independent biological replicates). P -values in c are from one-way ANOVA. RNA-seq analyses presented in d-i were from n = 4 independent biological replicates. Adjusted P -values used for d-i calculated by DESeq2 using the two-tailed Wald test with Benjamini-Hochberg correction.

Article Snippet: Small RNA sequencing with non-targeting mus DNMT1 ( mDNMT1 ) +6 G-to-C pegRNA or epegRNA (tevopreQ 1 ) was performed in quadruplicate, and for each replicate, 5 × 10 6 K562 PEmax parental or La-ko4 cells were nucleofected with 5,000 ng pegRNA or epegRNA plasmid using the SE Cell Line 4D-Nucleofector X kit L (Lonza, V4XC-1024) and pulse code FF120, according to the manufacturer’s protocol.

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Flow Cytometry, RNA Sequencing Assay, Expressing, Two Tailed Test