Search-and-Replace Genome Editing without Double-Strand Breaks or Donor DNA

• p. 2 Introduction, second paragraph

  such as the T•A-to-A•T mutation needed to directly correct the most common cause of sickle cell disease (HBB E6V)

• p. 10 Results, Prime editing pathogenic mutations

  Sickle cell disease is caused by a A•T-to-T•A transversion mutation in HBB

• p. 39 Figure 5 legend, panel (a)

  Installation (via T•A-to-A•T transversion) and correction (via A•T-to-T•A transversion) of the pathogenic E6V mutation in HBB

• p. 24 Extended Data Figure 4 legend, panel (g)

  (g) a +2 T•A-to-A•T transversion edit at HBB

Only the authors can say what is right here.

Judge's note: Read crops C001-a (p.2), C001-b/C001-f (p.10), C001-c/C001-g (p.39 Fig. 5 legend), C001-d plus the full page image main-p024.png (Extended Data Fig. 4 legend and panel g, whose plot title reads "HBB +2 T to A"). The texts are as quoted. Installation and correction are opposite directions of one change, so the Introduction (T•A-to-A•T = correcting) and the Fig. 5a legend (T•A-to-A•T = installing) use the notation in opposite senses; the paper nowhere states which strand the first-named base pair refers to, and the protospacer/PAM-strand convention implied by the "+2" position in Extended Data Fig. 4g is never declared for the main text. Downgraded to S3 because each statement can be internally consistent under a different reference strand (coding strand in the text, protospacer strand in the figure legends); only the authors can state the convention. The p.10 sentence defers to a Supplementary Discussion, which is not among the files.

Second check: another model agreed

• p. 11 Discussion and future directions, second paragraph

  Because 85-99% of insertions, deletions, indels, and duplications in ClinVar are ≤30 bp in length (Extended Data Fig. 11)

• p. 31 Extended Data Figure 11, panels a-d (x axes)

  Insertion length (bp)

Judge's note: Read crop C009-a (p.11) and the full Extended Data Fig. 11 on main-p031.png. All four panels (a insertions, b duplications, c deletions, d indels) plot length on an x axis running 0, 5, 10, 15, 20, 25, with the plot box closing at the 25 tick, and a right-hand cumulative frequency axis. The cited 30-bp threshold therefore cannot be read off the figure, although the legend makes clear the lengths were computed from the downloaded ClinVar variant summary, from which the authors could have taken the ≤30 bp fraction. Minor: the figure supports the length distribution but not the specific threshold quoted.

Second check: another model agreed

• p. 16 Methods, Determination of PE3 activity at known Cas9 off-target sites

  Variants that did not meet the criteria of p-value > 0.05 were excluded.

• p. 16 Methods, same paragraph

  to determine if the variants occurred at significantly higher levels compared to samples treated with pegRNAs that contained the same spacer but encoded different edits

• p. 16 Off-target methods

  Variants that did not meet the criteria of p-value > 0.05 were excluded

Only the authors can say what is right here.

Judge's note: Read crops C014-a/b/c and the full page main-p016.png; the sentence is printed exactly as quoted, with the ">" sign visible on the image. Taken literally, variants with p ≤ 0.05 (the significantly elevated ones the test was set up to find) would be excluded and off-target activity computed from the non-significant reads, which is the opposite of the stated aim. Rated S3 rather than S1 because the sentence is ambiguously constructed ("the criteria of p-value" followed by the threshold) and the intended direction is not recoverable from the page; only the authors can state the rule applied.

Second check: another model agreed

• p. 19 Methods, RNA-sequencing and data analysis

  Differentially expressed genes were called with FDR-corrected p-value < 0.05 and fold-change > 2 cutoffs

• p. 29 Extended Data Figure 8 legend, panels (c-h) and (i-k)

  Red dots indicate genes that show ≥2-fold change in relative abundance that are statistically significant

Judge's note: Read crops C017-a (p.19 Methods, RNA-sequencing and data analysis) and C017-b (p.29 Extended Data Fig. 8 legend). Both wordings are present as quoted on the page images; the legend uses the ≥ symbol twice (panels c-h and the Venn diagrams i-k) for the same limma-voom analysis the Methods describe. No statement elsewhere on these pages reconciles the strict versus inclusive form. The practical difference on continuous fold-change values is negligible, so this is a wording/definition ambiguity rather than a data error.

Second check: another model agreed

• p. 36 Figure 2 legend, panel (a)

  an engineered M-MLV reverse transcriptase (D200N, L603W, T306K, W313F, T330p)

• p. 5 Results, Prime editor 2 (PE2)

  M-MLV RT D200N+L603W+T330P+T306K+W313F

• p. 36 Figure 2 legend, panel (a)

  (D200N, L603W, T306K, W313F, T330p)

• p. 5 Results, "Prime editor 2 (PE2)"

  Cas9 H840A–M-MLV RT D200N+L603W+T330P+T306K+W313F

Judge's note: Read crop C027-a/-c/-e and crop C027-b of p.5. The Fig. 2a legend on the page image reads "(D200N, L603W, T306K, W313F, T330p)" with a lower-case p, while the Results on p.5 read "Cas9 H840A–M-MLV RT D200N+L603W+T330P+T306K+W313F" with an upper-case P, and the other four substitutions in the same parenthesis use upper-case residue letters. Visible in the rendered page image, so not an extraction artefact. Cosmetic.

Second check: none (judge only)

• p. 8 Results, Off-target prime editing, first sentence

  Prime editing requires target DNA:pegRNA spacer complementary for the Cas9 domain to bind

Judge's note: Read crop C005-a and the full page main-p008.png. The word is typeset as "complementary" in the first of the three parallel clauses, while the second and third read "target DNA:pegRNA PBS complementarity" and "target DNA:RT product complementarity". Not an extraction artefact: the page image shows the shorter word.

Second check: none (judge only)

• p. 14 Methods, General mammalian cell culture conditions

  HEK293T (ATCC CRL-3216), U2OS (ATTC HTB-96), K562 (CCL-243), and HeLa (CCL-2) cells were purchased from ATCC

• p. 14 General mammalian cell culture conditions

  U2OS (ATTC HTB-96)

Judge's note: Read crops C010-a and C010-b and the surrounding Methods on main-p014.png. The page image clearly shows "ATTC HTB-96" while the same sentence prints "ATCC CRL-3216" and "purchased from ATCC" (and later "EMEM, ATCC"), so the inconsistency is settled from internal evidence alone; no outside knowledge needed. Cosmetic misspelling with an evident fix.

Second check: none (judge only)

• p. 15 Methods, Nucleofection of U2OS, K562, and HeLa cells

  800ng prime editor-expression plasmid, 200ng PEgRNA-expression plasmid

Judge's note: Read crop C011-a and the full page main-p015.png. The typeset text reads "200ng PEgRNA-expression plasmid" in the "Nucleofection of U2OS, K562, and HeLa cells" Methods paragraph, whereas the same paragraph and the rest of the paper use "pegRNA" (e.g. "pegRNA or sgRNA plasmid" on p.16). Cosmetic.

Second check: none (judge only)

• p. 23 Extended Data Figure 3 legend, panel (d)

  using pegRNAs containing 13-nt Rt templates

• p. 23 Extended Data Figure 3 legend, panel (e)

  a pBs sequences ranging from 8-17 nt

• p. 23 Extended Data Figure 3 legend, panel (f)

  pegRNAs containing 11 -nt RT templates

• p. 23 Extended Data Fig. 3 (d),(e)

  13-nt Rt templates

Judge's note: Read crops C020-a/-b/-c/-d/-e/-h and the rendered page text. On the page image the (d) clause reads "13-nt Rt templates", the (e) clause reads "a pBs sequences ranging from 8-17 nt", and the (f) clause reads "11 -nt RT templates" with a space before the hyphen; the adjacent clauses (c), (e) first half and (g) use "RT templates" and "a PBS sequences". These are present in the typeset page, not an extraction artefact. Purely cosmetic. (The mis-capitalisations in the Extended Data Fig. 8/9/10 legends raised by notes 3-4 of this cluster are adjudicated separately as C022 and C024.)

Second check: none (judge only)

• p. 29 Extended Data Figure 8 legend, panel (c)

  comparing (c) PE2 vs. pE2-dRT with PRNP-targeting pegRNA

• p. 29 Extended Data Figure 8 legend, panel (f)

  (f) PE2 vs. PE2-dRT with HeXa-targeting pegRNA

• p. 29 Extended Data Figure 9 legend, panel (b)

  in HEK293T cells with pE3 or PE3b

• p. 29 Extended Data Fig. 8 legend

  PE2 vs. pE2-dRT

Judge's note: Read crops C022-a/-c/-d/-e/-f (and C017-b, which shows the whole Fig. 8 legend paragraph). On the page image the Fig. 8 legend prints "pE2-dRT" in the (c) clause while (d), (e), (f), (h), (i), (k) print "PE2-dRT"; it prints italic "HeXa" in the (f) clause while (g) and (h) print "HEXA". The Fig. 9 legend prints "with pE3 or PE3b" while the figure title and panel (a) print "PE3". Present in the typesetting, not extraction. Cosmetic only; the meaning is clear.

Second check: none (judge only)

• p. 31 Extended Data Figure 10 legend, panel (g)

  a +1 CTT insertion at HeK3 using Cas9-initiated HDR

• p. 31 Extended Data Figure 10 legend, panel (g)

  a +1 CTT insertion at HEK3 using PE3

Judge's note: Read crop C024-a and the full page 31 image. The sentence prints italic "HEK3" twice ("The reference HEK3 sequence...", "a +1 CTT insertion at HEK3 using PE3") and then "HeK3" in the immediately following clause. Present in the typeset page, so not an extraction artefact. Cosmetic.

Second check: none (judge only)

• p. 31 Acknowledgements

  P.J.C. and A.R. acknowledge an NSF graduate fellowships.

Judge's note: Read crop C025-a and the full page 31 image. The sentence is typeset as quoted; parallel sentences in the same paragraph read "acknowledges a Jane Coffin Childs postdoctoral fellowship", "acknowledges a Damon Runyon Cancer Research Foundation fellowship" and "acknowledges a Helen Hay Whitney postdoctoral fellowship". Singular article with plural noun; purely cosmetic.

Second check: none (judge only)

• p. 31 Extended Data Figure 11 title

  Extended Data Figure 11∣.

Judge's note: Read crop C026-a and the full page 31 image: the bold title shows "Extended Data Figure 11|." with a bar before the period. Compared with the other Extended Data titles on the page images (p.26 "Extended Data Figure 6.", p.27 "Extended Data Figure 7.", p.30 "Extended Data Figure 10."), which carry a plain period, so the character is an anomaly in this one title. Cosmetic typesetting only. (The 'a PBS sequences' grammar point mentioned in the same reviewer note belongs to the Extended Data Fig. 3 legend and is covered by C020.)

Second check: none (judge only)