Targeted genomic translocations and inversions generated using a paired prime editing strategy

Abstract

A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.

Keywords: CRISPR-Cas9; chromosomal inversion; chromosomal translocation; genome editing; genome rearrangement; prime editing.

Graphical abstract

Figure 1

PETI-mediated translocation in an episomal fluorescent reporter (A) Overview of the PETI method for generating episomal DNA recombination in human cells. The paired pegRNAs target the HEK3 and HEK4 sites that are incorporated into the two plasmids that make up the episomal fluorescent reporter system. PETI induces the formation of 3′-flaps at each of the target sites, which are complementary to sequences at the other target site, resulting in episomal translocations. (B) Representative fluorescence microscope images showing the absence or presence of eGFP expression, which results from episomal translocations potentially induced by PE2, PE2 nuclease, or Cas9 nuclease. Scale bar represents 100 μM. (C) Effects of 3′-flap length on translocation outcomes. Cells were transfected with plasmids encoding PE2, PE2 nuclease, or Cas9 nuclease and pegRNAs that generated 3′-flaps of different lengths (0–35 bp, increasing in increments of 5 bp), and the percentage of GFP-positive cells was evaluated using fluorescence-activated cell sorting. (D) Effects of combinations of pegRNA and gRNA on translocation outcomes. Cells were transfected with plasmids encoding PE2, PE2 nuclease, or Cas9 nuclease and pegRNA or gRNA pairs, and the GFP-positive cells were evaluated using fluorescence-activated cell sorting. (E) Cells were transfected with plasmids encoding Cas9 nuclease with gRNAs or PE2 nuclease with two types of pegRNA pairs (direct junction guided or 3 bp insertion guided), and the GFP-positive cells were evaluated using fluorescence-activated cell sorting. Data are presented as mean ± SEM (n = 2 biologically independent samples). (F) The relative frequency of sequence reads of amplicons generated using translocation-specific primers. Direct translocation and PETI-guided translocation (3 bp insertion) are shown in blue and red bars, respectively. (G) Results of targeted sequencing of amplicons generated using translocation-specific primers.

Figure 2

PETI-mediated translocation at an endogenous genomic locus (A) Representative schematic showing PETI-mediated translocations at endogenous target sites. PETI induces the formation of 3′-flaps at each of the target sites, which are complementary to sequences at the other target site, resulting in translocations. (B) Targeted sequencing of amplicons generated using translocation-specific primers. The bar charts represent the fraction of reads with direct translocations (blue), translocations with the intended insertion (orange) or deletion (green), and translocations with unwanted indels (gray). (C and D) Representative results of relative frequency of sequence reads in PCR amplicons amplified by translocation-specific primers, either H3F (HEK3 forward) and H4R (HEK4 reverse) or H4F and H3R. Direct translocation and PETI-induced translocation are indicated by blue and red bars, respectively. (E) Schematic overview of multiplex PCR to detect PE2-, PE2 nuclease-, or Cas9 nuclease-mediated translocations. Un-translocated genomic DNA is amplified by primers T1F and T1R or T2F and T2R, the junctions of desired translocations are amplified by T1F and T2R or T2F and T1R, and the junctions of undesired translocations are amplified by T1F and T2F or T2R and T1R. (F) Fraction of sequencing reads of multiplex PCR amplicons generated from desired and undesired PE2-, PE2 nuclease-, or Cas9 nuclease-mediated translocations. Data are presented as mean ± SEM (n = 3 biologically independent samples).

Figure 3

Use of PETI to generate a cancer-associated translocation (A and B) Schematic of the chromosomes before and after the reciprocal translocations generating the NPM1-ALK (a) and KIF5B-ALK fusion (b). (C and D) Representative results of relative frequency of sequence reads in PCR amplicons amplified by translocation-specific primers amplifying either junction-1 or junction-2. (E) Targeted deep sequencing of amplicons generated using translocation-specific primers. The bar charts represent the fraction of reads with direct translocations (blue), PETI-guided translocations (orange), and translocations with unwanted indels (gray). (F) Fraction of sequencing reads of multiplex PCR amplicons generated from desired and undesired PE2-, Cas9 nuclease-, and PE2 nuclease-mediated translocations. Data are presented as mean ± SEM (n = 3 biologically independent samples).

Figure 4

Use of PETI to generate cancer-associated inversions (A) Schematic of chromosome 2 before and after the paracentric inversion generating the EML4-ALK fusion. (B) Schematic of EML4-ALK fusion variants. EML4-ALK variant 1 (V1), variant 2 (V2), and variant 3 (V3) are generated by fusion of EML4 exon 13 to ALK exon 20, EML4 exon 20 to ALK exon 20, and EML4 exon 6 to ALK exon 20, respectively. (C) Representative results of relative frequency of sequence reads in PCR amplicons amplified by inversion-specific primers amplifying either junction-1 or junction-2. (D) Targeted deep sequencing of amplicons generated using inversion-specific primers. The bar charts represent the fraction of reads with direct inversions (blue), PETI-guided inversions (orange), and inversions with unwanted indels (gray). (E) Fraction of sequencing reads of multiplex PCR amplicons generated from desired and undesired PE2-, Cas9 nuclease-, and PE2 nuclease-mediated inversions. (F) Sequence chromatogram of a region of the EML4-ALK fusion transcripts showing the correct EML4-ALK junctions. (G and H) Flow cytometry analysis to estimate the percentage of cells with EML4-ALK V2 rearrangement by Cas9 and PE2 nuclease. p values were derived from Student’s two-tailed t test. Data are presented as mean ± SEM (n = 3 biologically independent samples).