Multiplex Genome Engineering Using CRISPR/Cas Systems
2013 · paper · Biomedicine
Engineers type II CRISPR/Cas systems so RNA-guided Cas9 cuts specific genome sites in human and mouse cells. It also shows a nicking version supporting homology-directed repair, and simultaneous editing of several sites using multiple guides in one array.
1 finding
Reference or label The body text's top/bottom pointers into Fig. 2B are reversed: "we adapted a chimeric crRNA-tracrRNA hybrid (Fig. 2B, top)" and "mimic the natural crRNA:tracrRNA duplex (Fig. 2B, bottom)", whereas the artwork and the legend place the pre-crRNA:tracrRNA complex on top and the chimeric RNA at the bottom.
- p. 3 Results, second paragraph
we adapted a chimeric crRNA-tracrRNA hybrid (Fig. 2B, top) design recently validated in vitro (12)
- p. 3 Results, second paragraph (same sentence)
to mimic the natural crRNA:tracrRNA duplex (Fig. 2B, bottom)
- p. 7 Fig. 2 legend, panel B
(B) Schematic of the pre-crRNA:tracrRNA complex (top) showing hybridization between the direct repeat (gray) region of the pre-crRNA and tracrRNA. Schematic of a chimeric RNA design (12) (bottom).
- p. 7 Fig. 2, panel B artwork (sub-panel headings)
pre-crRNA + tracrRNA processing
Judge's note: On the p.7 page image, panel B's upper sub-panel is headed "pre-crRNA + tracrRNA processing (30 bp)" and shows pre-crRNA hybridised with tracrRNA; the lower sub-panel is headed "chimeric RNA" with the "guide sequence (20 bp)" label (also visible in crop C002-c). The Fig. 2 legend on the same page states "Schematic of the pre-crRNA:tracrRNA complex (top) ... Schematic of a chimeric RNA design (12) (bottom)." The p.3 text (crop C002-a/-b and page image) points to "Fig. 2B, top" for the chimeric hybrid and "Fig. 2B, bottom" for the natural duplex, i.e. the opposite of both the artwork and the legend. Both pointers name panel B explicitly, so the whole-figure reading is excluded.
Second check: another model agreed
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3 minor (typos and the like; most checked by the judge only)
Wording On p.2 the abbreviation is introduced for a class that includes the tracrRNA — "two noncoding CRISPR RNAs (crRNAs): trans-activating crRNA (tracrRNA) and a precursor crRNA (pre-crRNA) array" — while later usage sets crRNA against tracrRNA ("crRNA:tracrRNA duplex", p.3; "processed pre-crRNA:tracrRNA complex (crRNA)", Fig. 2 legend, p.7).
- p. 2 Introduction, second paragraph
as well as two noncoding CRISPR RNAs (crRNAs): trans-activating crRNA (tracrRNA) and a precursor crRNA (pre-crRNA) array
- p. 3 Results, second paragraph
to mimic the natural crRNA:tracrRNA duplex
- p. 7 Fig. 2 legend, panel C
processed pre-crRNA:tracrRNA complex (crRNA)
Judge's note: Read the page images for p.2, p.3 and p.7 plus crop C001-a and the Fig. 2 legend crop. The printed sentences are as quoted: the introductory definition makes "crRNAs" the superset (tracrRNA + pre-crRNA), whereas the Results and the Fig. 2 legend use "crRNA" only for the mature guide RNA paired with tracrRNA. A real terminological inconsistency, but no number or result is affected and "CRISPR RNAs" can be read loosely as RNAs encoded at the CRISPR locus, so this is a minor wording/ambiguity point rather than an error.
Second check: another model agreed
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Wording Results (p.3) put the mismatch-intolerant region at "single-base mismatch up to 11 bp 5′ of the PAM completely abolished genomic cleavage by SpCas9" while the Discussion (p.4) cites the same panel for "the 14-bp (12 bp from the seed sequence and 2 bp from PAM) target specificity (Fig. 3B)", with no statement reconciling 11 bp and 12 bp.
- p. 3 Results, specificity paragraph
single-base mismatch up to 11 bp 5′ of the PAM completely abolished genomic cleavage by SpCas9
- p. 4 Discussion, first paragraph
the 14-bp (12 bp from the seed sequence and 2 bp from PAM) target specificity (Fig. 3B)
- p. 4 Discussion, first paragraph (end of HDR section)
Given the 14-bp (12 bp from the seed sequence and 2 bp from PAM) target specificity (Fig. 3B) of the wild-type SpCas9
- p. 3 Specificity paragraph
single-base mismatch up to 11 bp 5′ of the PAM completely abolished genomic cleavage
Only the authors can say what is right here.
Judge's note: Read p.3 and p.4 page images and crops C003-a, C003-b/-c/-e/-f/-h (text is as quoted on both pages), and the Fig. 3 panel on the p.8 page image. Fig. 3A lists mismatch guides m1–m9 and then m11, m13, m15, m17; in the Fig. 3B gel only the three most PAM-distal mutants carry indel values (5.6, 7.5, 8.8%) beside the 9.7% control lane, so position 12 is not tested and the data bound the critical region between 11 and 13 bp. The two sentences are therefore not arithmetically contradictory (11 ≤ 12), which is why I downgrade from the reviewers' "wrong number" reading; but the paper attributes two different lengths to the same figure without saying that 12 bp is an interpolation, so the ambiguity is real and only the authors can say which they mean.
Second check: another model agreed
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Typo Fig. 1 legend (p.6): "Mammalian expression of human codon–optimized SpCas9 (hSpCas9) and SpRNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1 α) promoter" — singular subject "expression" with plural verb "were driven".
- p. 6 Fig. 1 legend, panel B
Mammalian expression of human codon–optimized SpCas9 (hSpCas9) and SpRNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1 α) promoter
Judge's note: Read the p.6 page image and crop C006-a; the legend is printed as quoted, so this is not an extraction artefact. The head noun of the subject is "expression" (singular), so "were driven" disagrees; the parallel clause in the same sentence, "tracrRNA and pre-crRNA array (DR-Spacer-DR) were driven by the U6 promoter", is correct. Purely cosmetic: the meaning is unaffected.
Second check: none (judge only)