DNA Targeting Specificity of RNA-Guided Cas9 Nucleases
Nature Biotechnology, 2013 · paper · Biomedicine
Characterizes how precisely the SpCas9 nuclease targets DNA in human cells, testing over 700 guide variants and over 100 predicted off-target sites. Mismatch tolerance depends on number, position and distribution; dosage can be titrated to reduce off-target effects.
4 findings
Reference or label Abstract states indel levels were measured "at >100 predicted genomic off-target loci in 293T and 293FT cells", but no 293T experiment is described anywhere else in the article; the Online Methods name only HEK 293FT and the hESC line HUES9.
- p. 1 Abstract
levels at >100 predicted genomic off-target loci in 293T and 293FT cells
- p. 7 Online Methods, 'Cell culture and transfection'
Human embryonic kidney (HEK) cell line 293FT (Life Technologies) was maintained in
Only the authors can say what is right here.
Judge's note: Read the abstract on main p001 (crop C001-a and full page) and the 'Cell culture and transfection' section on main p007 (crop C001-b): the only HEK line named is 293FT, plus HUES9 hESCs (p007, SURVEYOR section '293FT and HUES9 cells were transfected...'). A grep of extract/main.txt shows '293T' occurs exactly once in the whole article, in the abstract line 24, while 293FT/HUES9/hESC occur 14 times. The named cell population in the abstract is therefore unsupported by anything in the article; only the authors can say whether some deep-sequencing samples were in fact 293T. The supplementary information is not among the files, so a 293T experiment reported only there cannot be checked.
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Reference or label Online Methods (p8) cites "HEK 293FT cells plated in 96-well plates were transfected with Cas9 plasmid DNA and sgRNA PCR cassette 72 h before genomic DNA extraction (Supplementary Fig. 4)", but the article assigns Supplementary Fig. 4 to the pUC19 methylation/cell-free cleavage experiment and Supplementary Fig. 5 to the sgRNA PCR cassette.
- p. 8 Online Methods, 'Deep sequencing to assess targeting specificity', first sentence
sgRNA PCR cassette 72 h before genomic DNA extraction (Supplementary Fig. 4)
- p. 3 Results, methylation paragraph
M. SssI-methylated pUC19 as DNA targets (Supplementary Fig. 4a, b)
- p. 3 Results, specificity paragraph
using PCR and transfecting the resulting amplicons (Supplementary Fig. 5)
- p. 8 Methods, Deep sequencing to assess targeting specificity
before genomic DNA extraction (Supplementary Fig. 4)
Judge's note: Crops C013-a, C013-d and C013-e show the Methods sentence as quoted. On p3 (crops C013-b and C007-a) the article states 'Using either unmethylated or M. SssI-methylated pUC19 as DNA targets (Supplementary Fig. 4a, b) in a cell-free cleavage assay ... regardless of CpG methylation status in either the 20-bp target sequence or the PAM (Supplementary Fig. 4c)', exhausting panels a-c of Supplementary Fig. 4 with methylation content; and on p3 (crops C013-c, C013-f) 'we developed a simple sgRNA testing assay by generating expression cassettes encoding U6-driven sgRNAs using PCR and transfecting the resulting amplicons (Supplementary Fig. 5)'. The Methods pointer for the same PCR-cassette transfection therefore names the wrong supplementary figure; the evident fix is Supplementary Fig. 5. The supplement itself is not among the files, but the article's own assignments are explicit and mutually exclusive.
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Reference or label Online Methods (PDF p. 10) attributes the statistical method to reference 2: "Wilson score intervals2 were calculated for each sample", but reference 2 is "Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:233–239."
- p. 10 Online Methods, 'Sequencing data analysis and indel detection' (continued)
Wilson score intervals2 were calculated for each sample
- p. 10 Reference list, entry 2
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013; 31:233–239.
- p. 10 Reference list, ref 2
2. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
- p. 10 Online Methods, sequencing data analysis
Wilson score intervals2 were calculated
Only the authors can say what is right here.
Judge's note: Opened crop C016-a: the '2' is typeset as a raised superscript immediately after 'intervals', matching the citation style used elsewhere in the Online Methods (e.g. 'Northern blots were done as previously described1' on p. 8, which points to Cong et al.). It is not an exponent: the displayed Wilson formula that follows is set separately. Opened crop C016-b (page 10 reference list): entry 2 is Jiang et al. on RNA-guided editing of bacterial genomes, whose title contains nothing on binomial confidence intervals. I grepped the extracted text for the reference list range: it runs 1–29 (ref 29, Mahfouz et al., on p. 11) with no statistics-methodology entry and no ref 30, so the superscript cannot be a truncated higher number. Only the authors can supply the intended source, hence author_query.
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Reference or label Reference 28 (PDF p. 11) is printed as "28. Michaelis, Maud LM. Die kinetik der invertinwirkung. Biochemistry Zeitung. 1913; 49:333–369." — a malformed author field and a journal title that does not identify the cited work.
- p. 11 Reference list, entry 28
Michaelis, Maud LM. Die kinetik der invertinwirkung. Biochemistry Zeitung. 1913; 49:333–369.
- p. 11 Reference list, ref 28
28. Michaelis, Maud LM. Die kinetik der invertinwirkung. Biochemistry Zeitung. 1913; 49:333–369.
- p. 11 Ref. 28
Michaelis, Maud LM. Die kinetik der invertinwirkung. Biochemistry Zeitung.
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: that the 1913 paper 'Die Kinetik der Invertinwirkung', volume 49, pages 333–369, is by L. Michaelis and M. L. Menten and appeared in Biochemische Zeitschrift. What I can verify in the files: crops C018-a and C018-b show the entry exactly as quoted, with 'Michaelis, Maud LM' as the whole author string and 'Biochemistry Zeitung' as the journal. The author field is internally malformed on its face — every other entry in the list (refs 24–27 in the same crop) uses the 'Surname Initials' form, whereas this one gives a forename ('Maud') with no accompanying surname, so one author's surname is absent. Title, year, volume and page range match the intended source, so the entry is identifiable but not correctly stated; only the authors can confirm the intended form.
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10 minor (typos and the like; most checked by the judge only)
Reference or label Accession codes states "All raw reads can be accessed at NCBI BioProject, accession number SRP023129", labelling an SRP-prefixed accession as a BioProject identifier.
- p. 1 Accession codes
NCBI BioProject, accession number SRP023129
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: NCBI accessions beginning SRP are Sequence Read Archive study accessions, whereas BioProject records carry PRJNA-prefixed accessions. The page itself (crop C004-a, confirmed on full page main-p001.png) prints exactly 'NCBI BioProject, accession number SRP023129'. The accession number itself is given and usable, so this is a repository-label imprecision rather than a wrong datum; only the authors can say which record they intend.
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Reference or label Online Methods (p8) attributes the fusion-PCR barcoding schematic to Supplementary Fig. 5 — "by a fusion PCR method to attach the Illumina P5 adapters as well as unique sample-specific barcodes to the target amplicons (schematic described in Supplementary Fig. 5)" — whereas the Results (p3) assign Supplementary Fig. 5 to the U6-sgRNA PCR expression cassette and Supplementary Fig. 6 to the deep-sequencing amplicons.
- p. 8 Online Methods, 'Deep sequencing to assess targeting specificity'
to the target amplicons (schematic described in Supplementary Fig. 5)
- p. 3 Results, specificity paragraph
performed deep sequencing of the region flanking each target site (Supplementary Fig. 6)
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Judge's note: Crops C014-a and C013-a show the Methods sentence citing Supplementary Fig. 6 for amplification of the flanking region and then Supplementary Fig. 5 for the schematic of the same fusion-PCR/barcoding step. Crop C014-b (p3) reads 'transfecting the resulting amplicons (Supplementary Fig. 5). We then performed deep sequencing of the region flanking each target site (Supplementary Fig. 6)'. Supplementary Fig. 5 is thus used for two different procedures, and together with C013 the Methods citations look shifted by one. I cannot fully settle which figure holds the barcoding schematic because the supplementary information is not among the files; the supplement's Fig. 5 and Fig. 6 legends would settle it. Downgraded to minor for that reason.
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Wording Page 2 concludes "these data indicate that the tracrRNA tail is important for optimal SpCas9 expression and activity in vivo", although the supporting northern blot (Fig. 1c) measured guide RNA levels, not SpCas9 expression.
- p. 2 Results, tracrRNA tail
the tracrRNA tail is important for optimal SpCas9 expression and activity in vivo
Only the authors can say what is right here.
Judge's note: Crop C006-a gives the whole paragraph: 'We performed northern blot analyses for the guide RNA truncations and found increased levels of expression for the longer tracrRNA sequences, suggesting that improved target cleavage was at least partially due to higher sgRNA expression or stability (Fig. 1c).' The Fig. 1 legend on main-p013.png describes panel c as 'Northern blot analysis for the four sgRNA truncation architectures, with U1 as loading control' — no measurement of SpCas9 protein appears anywhere in Fig. 1. The summary sentence attributes 'expression' to SpCas9 rather than to the sgRNA; it may be loose shorthand for the SpCas9-sgRNA system, so this is a wording/ambiguity point for the authors rather than a wrong number.
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Wording Page 4 says "we selected 32 target sites within the EMX1 locus encompassing all 16 possible alternate PAMs with 2× coverage", but the 16 combinations include the canonical 5′-NGG, so only 15 are alternates.
- p. 4 Results, PAM requirement paragraph
we selected 32 target sites within the EMX1 locus encompassing all 16 possible alternate PAMs with 2× coverage
- p. 14 Figure 2d and legend
SpCas9-mediated indel frequencies at targets with all possible PAM sequences
Judge's note: Crop C011-a gives the sentence; Figure 2d on main-p014.png is a 4x4 grid with rows NA, NC, NG, NT (first 2 PAM bases) against columns A, C, G, T (3rd PAM base), i.e. all 16 second/third-position combinations including the NG/G cell, and the legend (crop C011-b) says 'SpCas9-mediated indel frequencies at targets with all possible PAM sequences ... Two target sites from the EMX1 locus were tested for each PAM', giving 16 x 2 = 32 sites. The count 16 and the count 32 are internally consistent; only the word 'alternate' is loose, since the set includes the reference NGG. Wording point rather than a wrong number.
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Typo Abstract prints "by means of singleguide RNAs (sgRNAs) to enable genome editing" — 'single-guide' is run together without a hyphen.
- p. 1 Abstract, second sentence
by means of singleguide RNAs (sgRNAs) to enable genome editing
- p. 1 Abstract, first sentence
by means of singleguide RNAs (sgRNAs)
Judge's note: Checked crops C002-a and C002-b and the full page image main-p001.png. The typeset abstract line reads 'by means of singleguide RNAs (sgRNAs) to enable genome editing' with 'singleguide' set as one word in mid-line, so this is not a de-hyphenation artefact of text extraction. Cosmetic only.
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Typo Page 2 prints "through the induction of DNA doublestranded breaks (DSBs)" — 'double-stranded' run together without a hyphen.
- p. 2 Results, tracrRNA truncation paragraph
through the induction of DNA doublestranded breaks (DSBs)
Judge's note: Crop C005-a shows the typeset line 'embryonic kidney (HEK) 293FT cells through the induction of DNA doublestranded breaks' with 'doublestranded' as a single word in mid-line (the line break falls after 'breaks'), so it is in the PDF and not an extraction artefact. Cosmetic only.
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Typo Figure 3 legend (PDF p. 16): "analyzed by deep sequencing from two biological replicas", where the same design is called 'replicates' elsewhere, e.g. Results (p. 3) "for two independent biological replicates".
- p. 16 Figure 3 legend
sequencing from two biological replicas
- p. 3 Results, specificity paragraph
for two independent biological replicates
Judge's note: Opened crop C020-a (Figure 3 legend, 'All indel frequencies are absolute and analyzed by deep sequencing from two biological replicas. Error bars indicate Wilson intervals (Online Methods).') and crop C020-b (Results p. 3, 'We then performed deep sequencing of the region flanking each target site (Supplementary Fig. 6) for two independent biological replicates.'). Both read as quoted on the page images. The Online Methods (p. 10) likewise says 'for each biological replicate'. This is a word-choice inconsistency for the identical quantity, with no effect on any number or on the meaning, so I downgrade it to a cosmetic observation rather than a substantive defect.
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Typo Page 4 prints "Although we have shown here that methylated DNA sequences can be cleaved, by SpCas9 further characterization of the implications of epigenetic factors on CRISPR editing efficiency are needed." — the comma falls after 'cleaved' instead of after 'SpCas9' (and the singular subject 'characterization' takes 'are needed').
- p. 4 Results, PAM paragraph, last sentence
methylated DNA sequences can be cleaved, by SpCas9 further characterization of
- p. 4 Results, PAM requirement paragraph (last sentence)
methylated DNA sequences can be cleaved, by SpCas9 further characterization of the implications of epigenetic factors on CRISPR editing efficiency are needed
- p. 4 para on NAG PAM
can be cleaved, by SpCas9 further characterization
Judge's note: Crops C010-a, C010-b and C010-c all show the same typeset lines, and the comma is plainly set after 'cleaved' with 'by SpCas9' beginning the next clause; this is not an extraction artefact. The intended reading ('cleaved by SpCas9, further characterization ... is needed') is obvious, so the defect is punctuation/agreement only.
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Typo Acknowledgments (PDF p. 10): "D.A.S. is an National Science Foundation pre-doctoral fellow" — indefinite article 'an' before the consonant-initial 'National'.
- p. 10 Acknowledgments
D.A.S. is an National Science Foundation pre-doctoral fellow
Judge's note: Opened crop C017-a; the words 'is an National Science Foundation pre-doctoral fellow' appear as typeset on the page image, so this is not a text-extraction artefact. Purely cosmetic grammar; evident fix is 'a'. (Note the same paragraph also writes 'an NIH Director's' and 'an NIH Transformative', which are correct before the initialism.)
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Typo Figure 1d legend (PDF p. 13): "(d) Both wild-type (WT) or nickase mutant (D10A) of SpCas9 promoted insertion of a HindIII site" — 'Both' paired with 'or'.
- p. 13 Figure 1d legend
Both wild-type (WT) or nickase mutant
Judge's note: Opened crop C019-a; the legend reads '(d) Both wild-type (WT) or nickase mutant (D10A) of SpCas9 promoted insertion of a HindIII site into the human EMX1 gene' as typeset. The correlative 'both ... or' is ungrammatical; evident fix is 'and' (or deleting 'Both'). Cosmetic only — the meaning, that both enzymes promoted insertion, is clear from the plural verb.
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