Genetic Screens in Human Cells Using the CRISPR-Cas9 System
Science, 2014 · paper · Biomedicine
Describes pooled genome-scale loss-of-function screens in human cells using a library of 73,000 CRISPR guide RNAs. Screens identified mismatch repair genes for 6-thioguanine resistance, TOP2A and CDK6 for etoposide, essential gene sets, and sequence motifs predicting guide efficiency.
3 findings
Substance "The library included 10 sgRNAs for each of 7033 genes" plus 84 ribosomal genes does not give the stated "7,114 genes"; Fig. 1G implies 7031 genes with 10 sgRNAs each.
- p. 2 last paragraph
The library included 10 sgRNAs for each of 7033 genes
- p. 2 last paragraph
consisting of multiple sgRNAs targeting 7,114 genes
- p. 8 Fig. 1G, Total row
7114
- p. 2 Results, last paragraph
multiple sgRNAs targeting 7,114 genes
Only the authors can say what is right here.
Judge's note: Viewed page 2 text and the Fig. 1G table on page 8. The table's non-ribosomal pools sum to 7031 genes and 70310 sgRNAs (exactly 10 per gene), and with 83 ribosomal genes the total is 7114 genes and 73151 sgRNAs, as printed. The text's 7033 + 84 = 7117. I found no statement in the article about genes shared between pools. Table S1 and the Methods are not among the files, so which figure is right is for the authors.
Second check: another model agreed
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Substance Text says "each of the 84 genes encoding ribosomal proteins" (p. 3) and "each of the 84 ribosomal genes" (p. 4), but Fig. 1G lists 83 genes in the ribosomal protein pool.
- p. 3 first line of page
each of the 84 genes encoding ribosomal proteins
- p. 4 paragraph beginning 'Finally, we sought'
each of the 84 ribosomal genes
- p. 8 Fig. 1G, row 'Ribosomal protein', column Genes
83
- p. 4 Results, sgRNA efficacy paragraph
all possible sgRNAs against each of the 84 ribosomal genes
Only the authors can say what is right here.
Judge's note: The Fig. 1G row on page 8 reads 'Ribosomal protein 83 2741', and the printed gene total of 7114 holds only with 83. The text gives 84 twice. The article offers no explanation, such as a gene counted in another pool. The 'Fig. 1H' pointer also raised here is covered by C007.
Second check: another model agreed
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Reference or label "each of the 84 genes encoding ribosomal proteins (Fig. 1H)": Figure 1 has only panels A to G; library composition is panel G.
- p. 3 first line of page (library composition sentence)
each of the 84 genes encoding ribosomal proteins (Fig. 1H)
- p. 8 Fig. 1 and its legend
(G) Composition of genome-scale sgRNA library.
- p. 2 p2 library description
7,114 genes
- p. 2 p2
10 sgRNAs for each of 7033 genes and all possible sgRNAs for
Judge's note: The page 8 figure and its legend end at panel G ('Composition of genome-scale sgRNA library'); there is no panel H. The pointer should evidently be Fig. 1G. This cluster also carries the 7033 + 84 sum and the 84 vs 83 ribosomal count; those are judged under C002 and C008.
Second check: another model agreed
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8 minor (typos and the like; most checked by the judge only)
Reference or label "filtered for potential off-target effects based on sequence similarity to the rest of the human genome (Fig. 1, F and G)": panel G is the library composition table; the design and off-target filtering are shown in panels E and F.
- p. 2 last paragraph, library design sentence
based on sequence similarity to the rest of the human genome (Fig. 1, F and G)
- p. 8 Fig. 1 legend
(E) sgRNA library design pipeline. (F) Example of sgRNAs designed for PSMA4.
- p. 2 Results, last paragraph (library design)
off-target effects based on sequence similarity to the rest of the human genome (Fig. 1, F and G)
- p. 2 Results text
(Fig. 1, F and G)
Only the authors can say what is right here.
Judge's note: Viewed pages 2 and 8. Panel E is the design pipeline (target specificity, positioning), panel F the PSMA4 example of constitutive-exon targeting, panel G the pool composition table. Panel F does support half of the sentence, but G shows nothing about design or filtering. With the nonexistent 'Fig. 1H' in the next sentence, a one-letter shift (E and F intended) is likely, though only the authors can say.
Second check: another model agreed
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Wording "with tiny insertions or substitutions (<3 bp) occurring at a lower frequency": the Fig. 1D insertion histogram shows a visible share of insertions at 3 bp and at >3 bp.
- p. 2 Results, KBM7 AAVS1 paragraph
with tiny insertions or substitutions (<3 bp) occurring at a lower frequency
- p. 8 Fig. 1D, Insertion size histogram
1 2 3 >3
Judge's note: On page 8 the Fig. 1D insertion histogram has bars at 1 bp (about 0.7), 2 bp (about 0.05), 3 bp (about 0.2) and >3 bp (small). So '<3 bp' does not cover roughly a fifth of insertions; '≤3 bp' or loose wording may be intended. Bar heights are read by eye, so this is a wording observation only.
Second check: another model agreed
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Wording Discussion says "in the 6-TG screens" (plural) and "In the etoposide screen" (singular), whereas the results describe one 6-TG screen (Cas9-KBM7) and etoposide screens in both HL60 and KBM7.
- p. 5 Discussion, 'Third' paragraph
Moreover, in the 6-TG screens, the twenty most abundant sgRNAs
- p. 5 Discussion, 'Third' paragraph
In the etoposide screen, the two top genes scored far above background levels
- p. 3 Results, 6-TG and etoposide paragraphs
In both HL60 and KBM7 cells, we screened for genes whose loss conferred resistance to etoposide
Judge's note: Page 3 describes a single 6-TG screen in Cas9-KBM7 cells and etoposide screening 'in both HL60 and KBM7 cells' ('both screens'). Page 5 reverses the singular and plural. This is loose wording with no effect on any number.
Second check: another model agreed
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Typo "Mutant cells lines and mice bearing" (p. 2) and "strongly correlated between the two cells lines" (p. 4): 'cells lines' should be 'cell lines'.
- p. 2 second paragraph, last sentence
Mutant cells lines and mice bearing
- p. 4 Results, non-ribosomal genes paragraph
strongly correlated between the two cells lines
- p. 4 para before ribosomal gene analysis
correlated between the two cells lines
Judge's note: Both instances are visible on the page images of pages 2 and 4.
Second check: none (judge only)
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Typo "loss of function screening method in mammalian cells" ends the paragraph with no full stop.
- p. 2 end of first (continued) paragraph
of function screening method in mammalian cells
- p. 2 end of first paragraph
loss of function screening method in mammalian cells
Judge's note: On the page 2 image the first paragraph ends at 'mammalian cells' with no terminal punctuation.
Second check: none (judge only)
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Typo "We began by analyzing ribosomal proteins genes" and "majority of ribosomal proteins genes are essential": should be 'ribosomal protein genes'.
- p. 4 second paragraph, first sentence
We began by analyzing ribosomal proteins genes
- p. 4 paragraph beginning 'Finally, we sought'
ribosomal proteins genes are essential
- p. 4 para beginning "We began by analyzing"
analyzing ribosomal proteins genes
Judge's note: Both instances are visible on the page 4 image; the same page also uses 'ribosomal protein genes'.
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Typo "a fraction greater the next 400 sgRNAs combined": the word 'than' is missing.
- p. 5 paragraph beginning 'Third, off-target effects'
a fraction greater the next 400 sgRNAs combined
Judge's note: The page 5 image shows the wording as quoted, so it is not an extraction dropout.
Second check: none (judge only)
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Typo Fig. 3G legend (continued on page 12): "sgRNAs were designated as ‘weak’ or ‘strong’ based their log2 fold change" — the word "on" is missing ("based on their log2 fold change").
- p. 12 Fig. 3 legend, panel G (continued)
based their log2 fold change
Judge's note: Read the crop of page 12. The image shows "based their log2 fold change" with no "on", so this is not an extraction artefact. Cosmetic missing word with an evident fix.
Second check: none (judge only)