Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line

Abstract

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

Keywords: TP53 gene mutation; acute lymphoblastic leukemia; genome editing; prime editing system; treatment resistance.

FIGURE 1

Strategy to introduce R248Q mutation into the TP53 gene by the prime editing (PE)3b system. (A) Illustration of prime editing guide RNA (pegRNA). The original genomic DNA is indicated in black, pegRNA is indicated in blue. Targeted codon 248 is highlighted in yellow, and the protospacer adjacent motif (PAM) site is underlined in green. In pegRNA, the reverse transcriptase template (RTT) sequences are indicated in green, mutated nucleotides are in red. Scissor indicates breakpoint by nickase cas9. (B) Illustration of pegRNA and single guide RNA (sgRNA) (PE3b system). At the top of the panel, sequences of PBS and RTT in the pegRNA are indicated. RTT sequences are indicated in green, and mutated nucleotides are in red. In the middle, the original DNA sequence is indicated. Targeted codon 248 is highlighted in yellow. At the bottom, the spacer sequence of the pegRNA and the sgRNA are indicated. PAM sites are underlined in green. Scissors indicate breakpoints by nickase cas9. (C) Experiment workflow. (D) Dose–response curves of nutlin‐3a sensitivities in the subline and acute lymphoblastic leukemia parental cells. Horizontal and vertical axes indicate the concentrations of nutlin‐3a and cell viability in the alamarBlue assay, respectively. Error bars indicate SD of triplicate analyses. IC₅₀ value of the parental cells is indicated. *p < 0.001, t‐test.

FIGURE 2

Transactivation activity and downstream target gene expression of p53 protein. (A) Reporter assay of p53 protein. Top panel: sequences of WT and mutated p53 responsive elements in response element (RE; upper) and disrupted response element (dRE; lower) reporter plasmids are indicated. Bottom panel: relative luciferase (Luc) activities in acute lymphoblastic leukemia parental cells (left) and the subline (right) cultured in the presence (+) or absence (−) of 5 μM nutlin‐3a for 6 h. Luminescence produced by Renilla luciferase was used as an internal standard. Error bars indicate SD in triplicate analyses. BAS, basic; CTL, pGL3‐control; dRE, pGL3‐dRE; RE, pGL3‐RE. (B) Changes in the gene expression levels of CDKN2A (upper panel) and PUMA (lower panel). Real‐time RT‐PCR analysis was carried out in parental cells (left) and the subline (right) cultured in the presence or absence of nutlin‐3a at 5 μM for the indicated periods using the ACTB gene expression level as an internal control. Error bars indicate SD in triplicate analyses.

FIGURE 3

Drug and radiation sensitivities. (A, B) Dose–response curves to (A) vincristine and (B) L‐asparaginase in acute lymphoblastic leukemia parental cells and the subline. Horizontal and vertical axes indicate the concentrations of each drug and cell viability in the alamarBlue assay, respectively. IC₅₀ values are indicated in the graph. Error bars indicate SD in triplicate analyses. *p < 0.001, t‐test. (C) Radiation sensitivities of the parental cells and the subline. Vertical axes indicate cell viability (left) and living cell count (right) after 10 Gy radiation, while horizontal axes indicate the time after irradiation. Error bars indicate SD in triplicated analyses. *p < 0.001, t‐test.

FIGURE 4

Introduction of R248Q mutation with unintended editing errors by the prime editing (PE)3b system. (A) Sanger sequencing of the genomic PCR products from the acute lymphoblastic leukemia parental cells (middle panel) and the subline (lower panel). Top panel shows the original DNA sequence. Target codon 248 is highlighted in yellow. Red arrowheads indicate designed nucleotide substitutions, black arrowheads indicate unintended additional mutations. Scissors indicate breakpoint by nickase cas9. (B) Top panel: sequence of prime editing guide RNA (pegRNA). Green, red, and blue capitals indicate reverse transcriptase template (RTT), designed mutations, and maximum integrated sequences of the scaffold, respectively. Underlines indicate palindromic sequences in the 3′ scaffold. Bottom panel: original DNA sequence and eight patterns of mutated sequences. Codon 248 is highlighted in yellow. Green and red capitals indicate the RTT sequence and designed mutations, respectively. Blue, pink, and brown capitals indicate the pegRNA scaffold sequences, possible insertion/deletions, and the remaining original genomic DNA sequences at the target site, respectively.

FIGURE 5

Targeted next‐generation sequencing (NGS) analysis of 697 and HEK293T acute lymphoblastic leukemia cells transfected with the prime editing (PE)2 and PE3b systems. (A) NGS reads of 697 cells transfected with PE2 (left panels) and PE3b (right panels) systems before (upper panels) and after (lower panels) nutlin‐3a selection. (B) NGS reads of HEK293T cells (B) transfected with PE2 (left panel) and PE3b (right panel) systems without nutlin‐3a selection. In the bar charts, red, green, gray, and blue boxes indicate the designed R248Q and silent mutations, the designed mutations with error sequence, error sequence only, and WT sequence, respectively.

FIGURE 6

Possible mechanism for error editing. The original genomic DNA is represented in black, prime editing guide RNA (pegRNA) is represented in blue. In pegRNA, the reverse transcriptase (RT) template is indicated in green, designed mutations are indicated in red. The remaining original genomic DNA sequence is indicated in brown. Scissors indicate breakpoints by nickase cas9. Break‐up of the hairpin structure could induce extension of RT into scaffold sequence. Double‐strand break (DSB) due to a nick in the unedited strand could induce nonhomologous end‐joining, which could result in the insertion of scaffold sequence and the overlapping of unedited original genomic DNA sequence with insertion/deletions (indels). The insertions of pegRNA scaffold sequences, indels, and the overlap of the original genomic DNA sequence are indicated in light blue, pink, and brown, respectively.