(E) Similar plot as D showing sgRNAs of the HD CRISPR library B associated with each phenotype group. 12915_2020_905_MOESM13_ESM.pdf (110K) GUID:?8F3415A0-CB06-464D-BB94-94D018FB4437 Additional file 14: Figure S9. remained per gene after pre-filtering and were considered for library design. (G) Phenotypic deviation of published sgRNA phenotypes targeting the same gene. For each gene the difference between the GenomeCRISPR effect scores of the sgRNAs with the smallest and the largest effect scores was calculated. This process was repeated for each library using only those sgRNAs included in that library. Guides selected for the HD CRISPR libraries A and B show a narrow phenotypic deviation in published screens from which they were selected. 12915_2020_905_MOESM1_ESM.pdf (964K) GUID:?0B941131-C089-4EBB-AE0D-8FD8A5EBE8F1 Additional file 2: Table S1. Annotated sgRNA sequences of the HD CRISPR Library. 12915_2020_905_MOESM2_ESM.xlsx (34M) GUID:?24E290B0-041F-4C0F-9381-D0D818C014A7 Additional file 3: File S1. sgRNA sequences of the HD CRISPR Library A. 12915_2020_905_MOESM3_ESM.fasta (3.6M) GUID:?A4E7746F-0438-4C7B-870F-A4684B8A9908 REV7 Additional file 4: File S2. sgRNA sequences of the HD CRISPR Library B 12915_2020_905_MOESM4_ESM.fasta (3.4M) GUID:?214C4038-0CC0-495D-8C68-15130104915C Additional file 5: Figure S2. Features and performance of the HDCRISPRv1 vector. (A) Composition of the lentiviral HD CRISPR sgRNA expression vector. (B) sgRNA cloning efficiency can be addressed upon transfection of the HDCRISPRv1 vector, since residual GFP stuffer in non-digested vector backbone leads to GFP expression (B.l) (and editing efficiency was directly compared in the haploid and diploid population of the same cell line. Non-edited samples of the respective cell lines served as a control. Lines represent the mean of three independent experiments for each condition. 12915_2020_905_MOESM7_ESM.pdf (58K) GUID:?8813ACD9-5FAA-45B3-8818-47EC566F9AE9 Additional file 8: Figure S4. Cloning quality control of the HD CRISPR library. (A) LTI-291 Distribution of sgRNA read counts for the HD CRISPR plasmid library preparations. Skew ratios were determined as the quotient of the top 10 quantile divided by the bottom 10 quantile. (B) FACS analysis of GFP expression upon transfection of the HD CRISPR Library A and B plasmid pools to address the presence of remaining GFP stuffer (n?=?3 for each condition). 12915_2020_905_MOESM8_ESM.pdf (49K) GUID:?91417789-8DFA-4CFB-8EEB-0D519F78C923 Additional file 9: Figure S5. Reproducibility of negative selection screens with the HD CRISPR library. (A) Scatter plots showing the reproducibility of sgRNA phenotypes across biological replicates in screens with the HD CRISPR library. Each column includes screens performed in a bulk cell population (left) or in selected single cell clones with high Cas9 activity (middle and right). The top and bottom rows include screens with the HD CRISPR sub-libraries A and B, respectively. (B) Boxplot representing the distribution of the differences of the maximal and the minimal log2 fold change of guides targeting the same gene in individual screens. For each gene the difference between the maximal and the minimal sgRNA log2 fold change was calculated. This process was repeated for both HD CRISPR sublibraries using the phenotypes derived from screens in bulk population and single cell clones. Guides targeting the same gene LTI-291 result in similar log2 fold changes with a median difference of the maximal and the minimal log2 fold change smaller 1 for all screens. (C) Precision-recall-curve analysis for reference core essential and nonessential gene sets (Hart et al., 2015, Hart et al., 2017) of screens conducted in the HAP1 Cas9 bulk population using either the HD CRISPR Library A or LTI-291 B and two published CRISPR screens conducted in HAP1 cells using either the TKOv1 or TKOv3 library (Hart et al., 2017) as a reference. (D) Hit calling of the HD CRISPR Libraries A and B in comparison with a CRISPR screen conducted in HAP1 cells by Hart et al. (2017) using the TKOv1 library. (E) Hit calling of the HD CRISPR Libraries A and B in comparison with a CRISPR screen conducted in HAP1 cells by Hart et al. (2017) using the TKOv3 library. PCC?=?Pearson Correlation Coefficient, SCC?=?Spearman Correlation Coefficient. 12915_2020_905_MOESM9_ESM.pdf (1.0M) GUID:?3D8E108A-87FB-43AA-958B-087E361B4265 Additional file 10: Table S3. BAGEL scores for individual genes in individual screens. 12915_2020_905_MOESM10_ESM.xlsx (2.2M) GUID:?A88014CA-433F-4A01-8298-16BF72F3CCBE Additional file 11: Figure S6. Hit detection in screens with the HD CRISPR library. (A) Number of hits determined using BAGEL  at a strict Bayes factor cutoff (BF? ?6) in different screens conducted with the HD CRISPR library. (B) Number of essential genes determined using MAGeCK RRA  at 5% FDR in different screens conducted with the HD CRISPR library. (C) Number of essential genes determined.