Precise genomic deletions using paired prime editing

Abstract

Current methods to delete genomic sequences are based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 and pairs of single-guide RNAs (sgRNAs), but can be inefficient and imprecise, with errors including small indels as well as unintended large deletions and more complex rearrangements. In the present study, we describe a prime editing-based method, PRIME-Del, which induces a deletion using a pair of prime editing sgRNAs (pegRNAs) that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair. PRIME-Del achieves markedly higher precision than CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb, with 1-30% editing efficiency. PRIME-Del can also be used to couple genomic deletions with short insertions, enabling deletions with junctions that do not fall at protospacer-adjacent motif sites. Finally, extended expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful for precise, flexible programming of genomic deletions, epitope tagging and, potentially, programming genomic rearrangements.

Figure 1.. Precise episomal deletions using PRIME-Del.

a. Schematic of Cas9/paired-gRNA deletion strategy. b. Schematic of PE3 strategy, wherein the Prime Editor-2 enzyme and gRNA complex induce a nick (denoted as a gap in the bottom DNA strand), even after the correct editing event. c. Schematic of PRIME-Del using pairs of pegRNAs that target opposite DNA strands. Each pegRNA encodes the sites to be nicked at each end of the intended deletion, as well as a 3’ flap that is complementary to the region targeted by the other pegRNA. d. Cartoon representation of deletions programmed within the episomally-encoded eGFP gene (not drawn to a scale). e. PRIME-Del-mediated deletion efficiencies and error frequencies (with or without intended deletion) were measured for 24-bp, 91-bp, and 546-bp deletion experiments in HEK293T cells (averaged over replicates; n = 5). Sequencing reads were classified as without indel modifications (“No editing”), indel errors without the intended deletion, indel errors with the intended deletion, and correct deletion without error. f. PRIME-Del-mediated deletion efficiency was measured for the 546-bp deletion experiment using three methods. Error bars represent standard deviation for three replicates. g. Insertion, deletion and substitution error frequencies across sequencing reads from 546-bp deletion experiment. Reads were aligned to reference sequence either without (top) or with (bottom) deletion. Plots are from single-end reads with collapsing of UMIs to reduce sequencing errors; also shown with additional replicates and error-class-specific scales in Supplementary Fig. 1e. Note that only one of the two 3’-DNA-flaps is covered by the sequencing read in amplicons lacking the deletion (labeled as ‘wild-type’). h. Insertion, deletion and substitution error frequencies across the amplicons from 546-bp deletion experiment after merging paired-end sequencing reads.

Figure 2.. Concurrent programming of deletion and insertion using PRIME-Del.

a. Schematic of strategy, with reverse complementary sequences corresponding to the intended insertion in purple. b. Conventional strategy for deletion with Cas9 and pairs of gRNAs. Potential deletion junctions are restricted by the natural distribution of PAM sites. c. Pairs of pegRNAs were designed to encode five insertions, ranging in size from 3 to 30 bp, together with a 546 bp deletion in eGFP. d. Estimated deletion efficiencies and indel error frequencies (with or without intended deletion) in using these pegRNA pairs to induce concurrent deletion and insertion in HEK293T cells (averaged over replicates; n = 3). e. Representative insertion, deletion and substitution error frequencies plotted across sequencing reads from concurrent 546-bp deletion and 30-bp insertion condition. Plots are from single-end reads without UMI correction. Note that only one of the two 3’-DNA-flaps is covered by the sequencing read in amplicons lacking the deletion (labeled as ‘wild-type’). f. The percentage of reads containing the programmed deletion that also contain the programmed insertion. Error bars represent standard deviation for at least three replicates.

Figure 3.. Precise genomic deletions using PRIME-Del.

a. Schematic of generation of the eGFP-integrated HEK293T cell line. b. Estimated deletion efficiencies and error frequencies in using PRIME-Del for concurrent deletion and insertion on genomically integrated eGFP in HEK293T cells. (n = 3) c. Representative insertion, deletion and substitution error frequencies plotted across sequencing reads from concurrent 546-bp deletion and 30-bp insertion condition on genomically integrated eGFP. Plots are from single-end reads without UMI correction. d. Cartoon representation of deletions programmed within the HPRT1 gene. e. Deletion efficiencies measured for the 118-bp and 252-bp deletion using either PRIME-Del or Cas9/paired-gRNA (abbreviated to Cas9) strategies in HEK293T cells, quantified using either the unique-molecular identifier-based sequencing assay (UMI) or the droplet-digital PCR (ddPCR) assay. Error bars represent standard deviation for three replicates. f. Representative insertion, deletion and substitution error frequencies plotted across sequencing reads from 118-bp deletion (left) and 252-bp deletion (right) at HPRT exon 1, using the Cas9/paired-gRNA strategy. Different error classes are colored the same as in (c). g. Same as (f), but for PRIME-Del strategy. h. Estimated deletion efficiencies and indel error frequencies for different deletions across the genome for PRIME-Del (left) and Cas9/paired-gRNA (right) methods (averaged over replicates; n = 3). UMI-based sequencing assay was used for quantification (except the GC-rich amplicon of FMR1*, where added DMSO interfered with the UMI-addition reaction). i. Deletion efficiencies measured for 1-kb and 10-kb deletions at HPRT1 using either PRIME-Del (left) or Cas9/paired-gRNA (right) with ddPCR-based assay in HEK293T cells. Error bars represent standard deviation for three replicates. j. Fraction of reads with precise deletion measured for the 1-kb and 10-kb deletion on HPRT1 gene with either PRIME-Del (left) or Cas9/paired-gRNA (right) using sequencing of the deletion amplicons. Error bars represent standard deviation for three replicates.

Figure 4.. Extending the editing time window enhances prime editing and PRIME-Del efficiency.

a. Schematic for stably expressing both Prime Editor-2 enzyme and pegRNAs via two-step genome integration. b-c. Editing efficiencies measured for the 118-bp and 252-bp deletions at genomic HPRT1 exon 1 using PRIME-Del (paired-pegRNA construct) or CTT-insertion using prime editing (single-pegRNA construct) in K562(PE2) cells (b) or HEK293T(PE2) cells (c), as a function of time after initial transduction of pegRNA(s). Error bars represent standard deviation for three replicates.

Figure 5.. Potential advantages of using PRIME-Del in various genome editing applications.

The PRIME-Del strategy can be used to program precise genomic deletions without generation of short indel errors at Cas9 target sequences. Precision deletion, combined with ability to insert a short arbitrary sequence at the deletion junction, may allow robust gene knockout of active protein domains without generating a premature in-frame stop codon, which can trigger the nonsense-mediated decay (NMD) pathway. PRIME-Del may also allow replacement of genomic regions up to 10 Kb base-pairs with arbitrary sequences such as epitope tags or RNA transcription start sites. Single-stranded breaks generated during PRIME-Del are likely to be less toxic to the cell, especially when multiple regions are edited in parallel, potentially facilitating its multiplexing.