Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates

Abstract

Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).

Extended Data Fig. 1.. LPET-mediated precision editing.

a, The levels of LPET and in vitro transcribed epegRNA were measured by qPCR after electroporation in 293T cells (n = 3). b, Diagram of LPET, MS2-less LPET, ssDNA, modified petRNA (LPET, −17), and unmodified petRNA. c, LPET and no-RT controls. HEK293T cells were electroporated with indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol), and FANCF LPET(+2), PRNP LPET (+0), IDS LPET (+0), RUNX1 LPET (+2), HBB LPET (+2). Editing was measured by deep sequencing (n = 3). Data and error bars indicate mean and s.d. of three independent biological replicates. d, Analysis of precise editing, imprecise editing, and scaffold incorporation events (FANCF) by CRISPResso2-prime editing mode. ‘MODIFIED’ represents reads containing unexpected insertions, deletions, or substitutions. e, Representative reads for FANCF and PRNP. Some deletions at FANCF are likely caused by short homology (underlined) near the nicking sites. Top five reads are shown.

Extended Data Fig. 2.. MMLV RT-mediated precision editing with different LPET modifications.

a, Various modifications in the LPET. Precision editing by LPET with additional chemically modified residues [including Locked Nucleic Acid (LNA, IN), 2’-F, 2’-O-me (mN), and 3’ phosphorothioate (PS)] as measured by deep sequencing in 293T cells (n = 3). rN = RNA; * = 3’ PS linker. All sequences are written from 5’ to 3’. Data and error bars indicate the mean and s.d. of three independent biological replicates. b, RT mediates precise editing by LPETs with an all-DNA RTT and PBS with no modifications, or with the indicated 3’-terminal modifications, as measured by deep sequencing (n = 3). Blue and purple letters denote DNA, RNA and 2′-O-methyl RNA, respectively.

Extended Data Fig. 3.. Phi29 with unmodified and modified DPETs and additional controls.

a, Precise editing mediated by Phi29. HEK293T cells were electroporated with indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol), and indicated DPET(+0), MS2-less DPET, MS2-less single stranded DNA (RTT+PBS), modified linear DPET(−17)), or un-modified (all-RNA) DPET. b, Precise editing mediated by Phi29 and pegRNA. Editing efficiency was measured by deep sequencing (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates; two-tailed unpaired Student’s t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Extended Data Fig. 4 |. Phi29 DPE with different DPET modifications.

a, Phi29-mediated precision editing with various modifications in the DPET (including locked nucleic acid, 2′-fluoro, 2′-O-methyl, and 3′ phosphorothioate) as measured by deep sequencing (n = 3). * = 3′ PS linker. All sequences are written from 5′ to 3′. Data and error bars indicate the mean and s.d. of three independent biological replicates. b, Phi29 mediates precise editing by DPETs with an all-DNA RTT and PBS with no modifications, or with the indicated 3′-terminal modifications, as measured by deep sequencing (n = 3). Blue and purple letters denote DNA, RNA and 2′-O-methyl RNA residues, respectively. Data and error bars indicate the mean and s.d. of three independent biological replicates.

Extended Data Fig. 5.. Incremental DNA replacement in the PBS/RTT at the PRNP site.

Precision editing with DNA replacement in the PBS/RTT with LPET/RT or DPET/Phi29. Data and error bars indicate the mean and s.d. of three independent biological replicates.

Extended Data Fig. 6:. LPETs and DPETs enable precise genome editing without an additional nicking sgRNA in multiple endogenous sites.

a-c, HEK293T cells were electroporated with indicated mRNAs (1 μg), pegRNA (100 pmol, PE2) and nicking sgRNA (100 pmol, PE3). LPET and DPET groups include mRNAs, sgRNA and nicking sgRNA (100 pmol), and LPET/DPET (FANCF +2, PRNP +0, RUNX1 +2). Data and error bars indicate the mean and s.d. of three independent biological replicates.

Extended Data Fig. 7.. Biotinylated LPETs and DPETs enable precise genome editing.

(a-c), LPET/RT and DPET/Phi29 editing in multiple cell lines, as measured by deep sequencing (n = 3). ND, not determined. “SNP” indicates the existence of a SNP in the HBB target region in the U2OS cell line, precluding comparative testing. d-e, Representative FACS plots in Fig. 2f. f, Diagram depicting mSA-RT and Phi29. The 5’-biotinylated LPET or DPET is recruited by mSA and anneals to the nicked DNA. g, Tests of bio-LPETs and bio-DPETs in an mCherry reporter line and at endogenous sites (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates.

Extended Data Fig. 8.. PoII5M DNA polymerase, MarathonRT and TGIRT editing with synthetic templates.

a, Diagram depicting PoII5M/DPET editing (left). MCP-tethered PoII5M binds to the DPET via MS2. The DPET anneals to the nicked DNA strand and serves as the template for PoII5M DNA polymerase. Data indicate precision editing (3nt substitution) at the FANCF locus with DPET (+2) and fully Ome-substituted PBS (right). b, Precise editing at the FANCF locus with MarathonRT and TGIRT. Editing efficiency was measured by deep sequencing on day 3 (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates.

Extended Data Fig. 9:. Off-target and cytotoxicity evaluation.

a, On-target and off-target editing efficiencies for FANCF. Data and error bars indicate the mean and standard deviation of three independent biological replicates. b, HEK293T cells were electroporated with indicated mRNA reagents or buffers. Cell viability was measured by the CellTiter-Glo assay (n = 3) on the indicated time points. Data and error bars indicate mean and s.d. of three independent biological replicates. Two-tailed unpaired Student’s t-test: *, P < 0.05.

Extended Data Fig. 10:. Precise editing for a 40-bp replacement and a 132-bp insertion.

a, Precise editing for a 40-bp insertion and 90-bp deletion at two AAVS sites (1615 and 1705) using LPET/DPET through mRNA nucleofection, followed by PCR amplification and agarose gel electrophoresis. b, Quantification of 40bp insertion in a by deep sequencing (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates, unless otherwise indicated. c, Diagram of the TwinPE by LPETs/DPETs for long insertion. d, Precise editing for a 132-bp insertion at the HEK3 site. Editing efficiency was measured by ddPCR (n = 2).

Figure 1.. Prime editing with chimeric LPET.

a, Diagram depicting the putative mechanism of LPET editing. MCP-tethered RT binds to LPET via an MS2 stem-loop. The LPET anneals to the nicked DNA strand and serves as template for RT. b, Dose-dependent precise editing (deep sequencing) with the LPET (+0 configuration, see below) at the FANCF editing site. HEK293T cells were electroporated with indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol), and LPET. c, Precise editing mediated by LPET (+2 configuration, see below), MS2-less LPET(+2) (with end modification), MS2-less ssDNA LPET (without end modification), linear LPET(−17) with end protection, and unmodified, all-RNA LPET (without end modification). d, Precise editing mediated by LPETs with different DNA replacements at the FANCF site (3nt substitution, denoted by bold letters). Left: template sequences (edited bases in bold). The “sgRNA only” sample was cells only treated with sgRNA as a negative control. Stars denote phosphorothioate bonds. Green hairpins denote MS2. Right: editing efficiency was measured by deep sequencing on day 3 (n = 3). Grey bars denote indels. e, Precise editing by LPETs with fully 2′-O-methyl PBS (+0) or with “+1” to “+4” DNA, as measured by deep sequencing (n = 3) (hereinafter referred to as OMe LPET). f, g, Precise editing by LPET (+0) and unmodified LPET at IDS and PRNP endogenous sites. Data and error bars indicate the mean and s.d. of three independent biological replicates. two-tailed unpaired Student’s t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.. Phi29 DPE.

a, Diagram depicting the putative mechanism of phi29 DPE. MCP-tethered phi29 binds to the DPET via MS2. The DPET anneals to the nicked DNA strand and serves as the template for phi29 polymerase. b, Precise editing (3-nt substitution) at the FANCF locus by phi29 DNA polymerase and the same panel of templates from Fig. 1c, again through mRNA nucleofection. Gray bars denote indels. c, Precise editing by DPETs with fully 2′-O-methylated PBS (+0) or with ‘+1’ to ‘+4’ DNA (the same in Fig. 1d). The ‘no polymerase control’ was treated with nCas9 mRNA, sgRNA, nicking sgRNA and DPET (+2). d, FANCF DPET (+2), PRNP DPET (+0), HBB DPET (+2) and IDS DPET (+0) were used with phi29 and as LPETs alongside RT in the MDA-MB-231 cell line. e, Editing with LPET/DPET (+0) in human IMR90 primary lung fibroblasts at the PRNP and IDS loci. For b–e, editing efficiency was measured by deep sequencing on day 3 (n = 3). f, LPET/RT- or DPET/ phi29-based editing restored mCherry expression by a 4-nt insertion in a reporter cell line. mCherry was quantified by flow cytometry (n = 3). For all graphs, cells were electroporated with the indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol) and LPET or DPET (100 pmol). Data and error bars indicate mean and s.d. of three independent biological replicates; Pol, polymerase. g, Precision genome editing using mSA-tethered polymerases and biotinylated templates. Biotinylated LPET/DPET are shown with different modification patterns. Asterisks denote phosphorothioate linkages. h,i, Precise editing (3-nt substitution) at the FANCF locus by mSA–RT/biotinylated LPET or mSA–phi29/ biotinylated DPET in HEK293T cells. The ‘sgRNA only’ sample was from cells treated only with sgRNA as a negative control. Editing efficiency was measured by deep sequencing (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates.