Cleavage-free human genome editing

Abstract

Most gene editing technologies introduce breaks or nicks into DNA, leading to the generation of mutagenic insertions and deletions by non-homologous end-joining repair. Here, we report a new, cleavage-free gene editing approach based on replication interrupted template-driven DNA modification (RITDM). The RITDM system makes use of sequence-specific DLR fusion molecules that are specifically designed to enable localized, temporary blockage of DNA replication fork progression, thereby exposing single-stranded DNA that can be bound by DNA sequence modification templates for precise editing. We evaluate the use of zinc-finger arrays for sequence recognition. We demonstrate that RITDM can be used for gene editing at endogenous genomic loci in human cells and highlight its safety profile of low indel frequencies and undetectable off-target side effects in RITDM-edited clones and pools of cells.

Keywords: cleavage-free and off-targets; genome editing; human cells.

Graphical abstract

Figure 1

RITDM gene editing strategy (A) A RITDM system consists of a programmable DLR molecule containing a sequence-specific recognition domain (D), such as a zinc-finger array, fused to a linker domain (L), and a non-sequence-specific DNA binding domain (R). (B) Schematic of RITDM editing. A locus-specific DLR molecule binds DNA at the target site to block replication fork progression temporarily, and a DNA correction template anneals and incorporates at the DNA replication fork. The resulting intermediary DNA heteroduplex can be repaired to enable a permanent genetic modification.

Figure 2

Genome editing by RITDM (A) Diagram of a mutated EGFP (EGFPDP2) reporter gene in the HEK293 reporter cell line. The EGFDP2 reporter has a deletion of single nucleotide (G) and a G-to-C point mutation. Restoration of EGFP reading frame after RITDM editing. (B) Schematic depicting that a mutated EGFP (EGFPDP2) reporter gene was targeted and repaired using a specifically designed RITDM system. The DLR molecule recognizes 15-nucleotide sequences, specifically. (C) EGFP was restored to express functionally in the reporter cells, as shown in forms of positive cells (left), cellular cluster (middle), and enrichment of positive cells (right). (D) Representative flow plots from flow cytometric analysis of alive EGFP cells 7 days post-genomic editing with the indicated conditions of control, donor alone, RITDM genome editing. Quantification of GFP (+) cells (per million) from the indicated conditions. Bar graphs represent two independent experiments with standard error shown. (E) The endogenous ApoE gene was genetically modified at codon 112 by RITDM in HEK293 cells. (F) The DLR molecule 27 nucleotide sequences at the target. (G) Single-nucleotide T-to-C conversion was detected by ddPCR. The bar graph represents the editing frequencies at codon 112 of the ApoE gene achieved by RITDM in HEK293 cells measured and calculated by ddPCR. (H) Enhanced genome editing frequencies were detected by ddPCR, while ssODN alone did not induce T-to-C conversion. The bar graph represented the editing frequencies at codon 112 of the ApoE gene achieved by RITDM in HEK293 cells measured and calculated by ddPCR.

Figure 3

Illustrations of the scope of genome editing by RITDM A number of disease-related genomic loci were modified by RITDM. For each case, the genomic target sequence is shown in blue and the sequence to be changed in red. The donor template with the desired sequence modification (in green) is shown. For each case the type of editing and the intended consequences are indicated. A representative ddPCR plot illustrates typical experimental data obtained, while the bar charts show the percentage of cells without indels, the percentage of cells edited as intended, respectively, the percentage of cells with indels, as determined by deep sequencing. Experimental results are shown for ApoE (A), Bcl11a (B), and PDCD1 (C). Bar graphs represent at least two independent experiments with standard errors shown.

Figure 4

RITDM editing of genomic DNA in human B lymphocytes RITDM editing efficiencies and indel frequencies achieved at two genomic sites in human B lymphocytes. We used RITDM to convert T to C at codon 112 of ApoE (A) in human B lymphocytes respectively to insert two nucleotides, “GA,” at the beginning of exon 51 of the Dystrophin gene (B) both in the U937 cell line and in human B lymphocytes. The bar charts show the percentages of deep-sequencing reads without indels (the intended 2-nucleotide insertion is scored as indel), with genomic edits as intended (2-nucleotide insertion), respectively, with unintended indels from pooled RITDM-edited cells. Editing efficiencies and indel frequencies from each independent experiment are shown. Results obtained from the various RITDM editing experiments in human B lymphocytes were calculated from all biological replicates and displayed as mean ± SD (n = 3 independent experiments for ApoE and n = 4 independent experiments for Dystrophin genomic editing by RITDM. ∗p < 0.05).

Figure 5

RITDM results in low indels at the target sites (A) Overall indel frequencies at the EGFPDP2 target site comparing control, positive, and negative cell populations. (B) Overall indel distribution over a 171-nucleotide window in positive and negative cell populations after RITDM gene editing. (C) Overall indel frequencies (mean ± SEM) at ApoE codon 112, comparing positive (n = 3) and negative clones (n = 2). (D) Indel frequencies in a 108-nucleotide window in representative positive and negative clones of ApoE codon 112 conversion by RITDM. The y axis scale maximum is 0.25%.

Figure 6

SIRF analysis showing the interaction between the DLR fusion protein and nascent replication forks (A) Schematic of detection of interaction of DLR with a replication fork by SIRF assay (in situ analysis of protein interactions at the DNA replication fork). (B). Arrows point to red puncta indicating DLR interactions with the replication fork.