--- name: bio-crispr-screens-base-editing-analysis description: Analyzes base-editing screens for variant function. Covers library design (Sanson 2020 GRACE, Hanna 2021 BRCA1/2 SNV scanning, Cuella-Martin 2021), CBE vs ABE chemistry choice (BE3/BE4 vs ABE7.10/ABE8.20/ABE8e), editing-window math (positions 4-8 from PAM-distal end, wider for ABE8e), bystander-edit quantification and the variant-call ambiguity it creates, sgRNA-efficiency filtering before hit calling, indel byproduct interpretation, the substitution-vs-indel diagnostic, variant annotation against ClinVar / COSMIC, and the Broad be-validation-pipeline. Use when designing a BE variant screen, choosing CBE vs ABE for a specific edit, interpreting bystander-confounded hits, distinguishing functional signal from indel artifact, integrating CRISPResso2 output with screen scoring, or deciding BE vs PE for SNV installation. tool_type: mixed primary_tool: CRISPResso2 --- ## Version Compatibility Reference examples tested with: CRISPResso2 2.2.14+, BE-Hive 1.0+ (BE prediction), pandas 2.2+, biopython 1.83+, numpy 1.26+, scipy 1.12+, scikit-learn 1.4+, Broad be-validation-pipeline 1.0+ (Python). Before using code patterns, verify installed versions match. If versions differ: - CLI: `CRISPResso --version`; `be-validation-pipeline --help` - Python: `pip show CRISPResso2 be-hive` If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying. ## Base Editing Screen Analysis **"Analyze my base-editor variant-function screen"** -> Quantify per-sgRNA target-base conversion, bystander rate, and indel byproducts from amplicon sequencing; filter on editing efficiency; map each sgRNA to its intended SNV (target + bystander pattern); compute per-variant fitness from the screen log-fold change; reconcile target vs bystander variant attribution; annotate against ClinVar / COSMIC. - CLI: `CRISPResso --base_editor_output` for per-amplicon BE quantification - CLI: Broad `be-validation-pipeline` for end-to-end pooled-screen analysis with editing-efficiency filtering - Python: `BE-Hive` (Arbab 2020) for editing-efficiency prediction - Python: `BE-Designer` (Hwang 2019) for variant-encoding sgRNA design ## Base Editor Chemistry Selection | Editor | Reaction | Editing window | Indel byproduct rate | When to use | |--------|----------|----------------|----------------------|-------------| | BE3 (Komor 2016) | C->T (also G->A on opposite strand) | Pos 4-8 from PAM-distal end | 5-10% | Original; superseded | | BE4 / BE4max (Koblan 2018) | C->T | Pos 4-8 | <5% | CBE standard | | eA3A-BE3 | C->T narrow specificity | Pos 5-7 | <5% | Specifically TC contexts (eA3A prefers TC) | | ABE7.10 (Gaudelli 2017) | A->G (T->C opposite strand) | Pos 4-7 | <2% | First ABE; slow at non-TA contexts | | ABE8.20 (Richter 2020) | A->G | Pos 4-8 | <2% | Modern ABE; high activity | | ABE8e (Lapinaite 2020) | A->G | Pos 4-8 | <2% | Highest editing activity; broader window | | evoCDA-BE | C->T (broader) | Pos 1-9 | 5-10% | Larger editing window; more bystander | | CGBE1 (Kurt 2021) | C->G | Pos 5-7 | 5-10% | C-to-G transversion; rare use | | GBE (Zhao 2021) | C->G or C->A | Pos 4-7 | 5-10% | Transversions; less mature | **Decision rule:** For a target SNV at position 4-8 of a candidate spacer with no bystander Cs/As in the same window, BE3-BE4 or ABE7.10 is sufficient. For high-throughput variant scanning where bystander tolerance must be minimized, use eA3A-BE3 (TC contexts only) or ABE8e (narrower effective window). ## Editing Window Math **Why this matters for postdoc-level use:** Base editors are tethered to dCas9 (or nCas9) and the deaminase acts on the displaced ssDNA "R-loop" formed when Cas9 binds. The deaminase has a fixed reach -- positions 4-8 from the PAM-distal end of the protospacer for canonical BE3/BE4/ABE7.10. Outside this window, editing efficiency drops by 10-50x. ``` PAM-distal end PAM-proximal | | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 NGG ^^^^^^^^^^^ Canonical editing window (positions 4-8) For BE4max / ABE7.10: positions 4-8 are 5-50x more efficient than positions 1-3 or 9-13 For ABE8e: window extends to positions 4-10 due to enhanced TadA8e activity For evoCDA-BE: window 1-9 (broader; more bystander) ``` **Critical implication for variant interpretation:** If the intended edit is at position 5 and there is an additional editable C/A at position 7, both will be edited in the same molecule. The screen scores the *combination* of edits, not the intended one alone. This is bystander confounding. ## sgRNA Library Design for BE Screens **Goal:** Tile editing-window-positioned spacers across a protein region of interest to enable variant scanning. **Approach:** For each amino acid in the target region, find NGG-adjacent spacers where the SNV-of-interest base falls in editing positions 4-8 with minimal bystander C/A in the same window. Annotate each spacer with the predicted amino acid changes (target + bystander). ```python import pandas as pd import re from Bio.Seq import Seq def find_be_spacers(cds_sequence, cds_protein_start, target_aa, target_base='C', editor='BE4max'): '''Find sgRNAs that place target_base in editor-specific window at target_aa. Returns spacers with bystander annotation. Args: cds_sequence: nucleotide CDS (translated frame 1) cds_protein_start: amino acid number of CDS start (usually 1) target_aa: amino acid number to install variant (e.g., 130 for residue 130) target_base: 'C' (CBE) or 'A' (ABE) editor: 'BE3', 'BE4max', 'eA3A-BE3', 'ABE7.10', 'ABE8.20', 'ABE8e', 'evoCDA-BE' Returns: DataFrame with spacer, position-in-cds, target-base-position-in-spacer, bystander_positions, predicted_aa_changes ''' # Editor-specific editing window (positions from PAM-distal end of spacer) window_by_editor = { 'BE3': (4, 8), 'BE4max': (4, 8), 'eA3A-BE3': (5, 7), 'ABE7.10': (4, 7), 'ABE8.20': (4, 8), 'ABE8e': (4, 10), # ABE8e wider! 'evoCDA-BE': (1, 9), } window_lo, window_hi = window_by_editor[editor] aa_index = target_aa - cds_protein_start # 0-indexed in protein aa_start_nt = aa_index * 3 # nt offset in cds candidates = [] spacer_len = 20 pam_pattern = re.compile(r'(?=([ACGT]GG))') for strand, seq in [('+', cds_sequence), ('-', str(Seq(cds_sequence).reverse_complement()))]: for pam_match in pam_pattern.finditer(seq): pam_pos = pam_match.start() spacer_start = pam_pos - spacer_len if spacer_start < 0: continue spacer = seq[spacer_start:pam_pos] # Editor-specific window from PAM-distal end (1-indexed) # Find all editable bases in window edit_bases_in_window = [] for i, b in enumerate(spacer[window_lo-1:window_hi], start=window_lo): if b == target_base: edit_bases_in_window.append(i) if not edit_bases_in_window: continue # Annotate which edits hit the target_aa codon target_codon_start = aa_start_nt target_codon_end = target_codon_start + 3 target_position_in_spacer = [] for i in edit_bases_in_window: genomic_pos = spacer_start + i - 1 if target_codon_start <= genomic_pos < target_codon_end: target_position_in_spacer.append(i) bystander_positions = [i for i in edit_bases_in_window if i not in target_position_in_spacer] candidates.append({ 'spacer': spacer, 'strand': strand, 'spacer_start': spacer_start, 'target_positions': target_position_in_spacer, 'bystander_positions': bystander_positions, 'n_bystanders': len(bystander_positions), }) return pd.DataFrame(candidates).sort_values('n_bystanders') ``` **Decision rule:** Select spacers with target_positions != empty AND n_bystanders minimized. For variant-by-variant scanning, accept up to 1-2 bystanders if biology of those positions is interpretable; flag for downstream variant attribution. ## Editing Efficiency Filtering (Critical Pre-Hit-Calling) **Goal:** Drop sgRNAs that do not edit efficiently, since unedited reads represent no biological perturbation. **Approach:** From CRISPResso2 output, compute target-base-conversion percentage per sgRNA; filter library to sgRNAs with >50% target editing in a pilot or co-screened control. ```python def filter_by_editing_efficiency(crispresso_outputs_dir, target_pos, target_base, efficiency_threshold=0.5): '''Drop sgRNAs that edit = efficiency_threshold}) return pd.DataFrame(results) ``` **Convention:** Drop sgRNAs below 50% editing for variant-function screens. Hanna 2021 used a 30% threshold for primary screen and a 50% threshold for confirmed hits. Below 30%, the screen has insufficient power; above 70%, results approach saturation editing. ## Bystander Edit Attribution **Why this matters:** When a sgRNA's editing window contains the target base AND a bystander base, the screen scores the combination. To attribute screen signal to the target variant alone, either (a) include sgRNAs that edit only the target (no bystander) -- often impossible -- or (b) deconvolute via parallel measurements. **Strategies for variant-by-variant attribution:** 1. **Tile multiple sgRNAs with different bystander patterns:** If 5 different sgRNAs all hit the target base but have different bystanders, common signal across them is target-attributable (Hanna 2021 approach). 2. **Use orthogonal chemistry:** Run the same variant scan with prime editor (no bystanders); cross-validate. See [[prime-editing-screens]]. 3. **Bystander stratification:** From CRISPResso2 allele table, partition reads by exact edit pattern (target only, target+bystander_1, target+bystander_2, etc.); separately score each pattern's contribution to the phenotype. 4. **Restrict library:** Use only sgRNAs with zero bystanders in the editing window (rare; may exclude most candidate spacers). ```python def deconvolute_bystander(allele_table_path, target_pos, bystander_pos_list): '''From CRISPResso2 allele table, partition reads by edit pattern at target + bystanders. Returns: per-pattern frequency for each combination of target/bystander edits.''' alleles = pd.read_csv(allele_table_path, sep='\t', compression='zip') # Mark target_edited and per-bystander_edited alleles['target_edited'] = alleles['Aligned_Sequence'].str[target_pos-1] != alleles['Reference_Sequence'].str[target_pos-1] for bp in bystander_pos_list: alleles[f'bystander_{bp}_edited'] = alleles['Aligned_Sequence'].str[bp-1] != alleles['Reference_Sequence'].str[bp-1] return alleles.groupby(['target_edited'] + [f'bystander_{bp}_edited' for bp in bystander_pos_list])['Reference_pct'].sum().reset_index() ``` ## Hit Calling for Variant-Function Screens **Goal:** Score per-variant fitness from a base-editor screen. **Approach:** Filter library to efficiency-passing sgRNAs (>50% editing), then run MAGeCK MLE or drugZ on the sgRNA-level counts; map each significant sgRNA to its predicted variant + bystander pattern; aggregate to per-variant scores. ```python def aggregate_variant_scores(mageck_sgrna_summary, variant_annotation_df): '''Aggregate sgRNA-level scores to per-variant scores. variant_annotation_df: per-sgRNA -> predicted variants (target + bystanders).''' df = mageck_sgrna_summary.merge(variant_annotation_df, on='sgRNA') # Target-only contribution: sgRNAs with no bystanders target_only = df[df['n_bystanders'] == 0] target_only_scores = target_only.groupby('target_variant')['LFC'].agg(['mean', 'std', 'count']) # Mixed signal: sgRNAs with bystanders mixed = df[df['n_bystanders'] > 0] return target_only_scores, mixed ``` ## Hanna 2021 BRCA1/2 Variant-Function Screen Methodology **Hanna et al 2021 *Cell* 184:1066** established the gold-standard methodology for BE variant scanning: 1. Design CBE library tiling BRCA1 and BRCA2 with 10-15 sgRNAs per amino acid 2. Verify editing efficiency in a control timepoint via amplicon sequencing 3. Drop sgRNAs <30% target editing 4. Run drug-modifier screens (PARPi sensitivity) with vehicle vs drug 5. Use drugZ to identify sensitizing variants (loss of function) 6. Per-variant scoring: aggregate over all sgRNAs that hit that variant; cross-check against bystander-controlled sgRNAs 7. Cross-validate hits via prime-editor scans of the same variants **Quantified result:** Identified novel loss-of-function variants in BRCA1 RING and BRCT domains, MCL1 and BCL2L1, and PARP1 resistance variants. The approach validated for clinical variant interpretation. ## Cuella-Martin 2021 DDR-Gene Variant Screening **Cuella-Martin et al 2021 *Cell* 184:1081-1097** screened ~86 DNA-damage-response (DDR) genes (including BRCA1/2) with CBE saturation mutagenesis: - Saturation CBE design across 86 DDR genes (not BRCA1/2 alone) - Identified pathogenic/likely-pathogenic variants in critical protein domains - Combined with proteomic validation - Demonstrated saturation mutagenesis is feasible at protein-domain scale **Reconciliation with Hanna 2021:** Both studies converged on similar functional variant calls in BRCA1 RING and BRCT domains; the orthogonal methodology gave confidence in clinical interpretation. ## Cas9 vs Base Editor vs Prime Editor for Variant Installation | Approach | What it does | Bystander | Indels | When to use | |----------|--------------|-----------|--------|-------------| | Cas9 + HDR template | Installs precise edit + template | None | High (NHEJ competition) | When precise edit needed; high indel byproduct | | Cas9 (no template) | Random indels at cut site | None | 70%+ | Loss-of-function; not variant-specific | | CBE (BE3/BE4) | C->T at editing window | Yes (multiple Cs) | <5% | C->T variants with manageable bystanders | | ABE (ABE7.10/ABE8e) | A->G at editing window | Yes (multiple As) | <2% | A->G variants; clean for single-A spacers | | CGBE / GBE | C->G or C->A | Yes | 5-10% | Transversions; rare use cases | | Prime editor (PE2/PE3) | Templated edit; any base change | None | 1-3% | Precise variants; lower efficiency | **Decision:** For C->T or A->G with available editing window: base editor is preferred (higher efficiency than PE). For other transitions/transversions, multi-base edits, or zero-bystander requirements: prime editor. ## Broad be-validation-pipeline The Broad Institute's `be-validation-pipeline` (https://broadinstitute.github.io/be-validation-pipeline/) is an end-to-end snakemake workflow for BE variant-function screens: ```bash # Install git clone https://github.com/broadinstitute/be-validation-pipeline cd be-validation-pipeline conda env create -f environment.yaml conda activate bevalidation # Configure edit config.yaml # Specify library, FASTQ paths, reference, target genes # Run snakemake --use-conda --cores 16 # Outputs: # results/per_sgrna_editing_efficiency.tsv # results/per_variant_attribution.tsv # results/hit_calls.tsv ``` The pipeline handles editing-efficiency filtering, bystander attribution, and variant calling in a single end-to-end workflow optimized for the GRACE library (Sanson 2020) but adaptable. ## Failure Modes ### Mostly indels in BE sample **Trigger:** Cas9 contamination, wrong vector (e.g., used pCas9-BE3 plasmid but selected on Cas9 line), or evoCDA-BE / broader-window chemistry. **Mechanism:** Cas9 cuts dsDNA; BE relies on nicked-ssDNA deamination. Cas9 expression in the same cell creates indels. **Symptom:** Substitution-vs-indel ratio <3 in CRISPResso output. **Fix:** Verify vector (nCas9-BE3 not Cas9-BE3); confirm cell line lacks Cas9 background; restrict to specifically engineered BE-cell lines. ### High editing but no biological signal **Trigger:** Bystander C/A is dominating; intended variant is not the perturbation driving phenotype. **Mechanism:** When target is at position 5 and bystander is at position 7, the molecule carries both; phenotype is from the bystander. **Symptom:** Strong screen signal but variant attribution unclear. **Fix:** Run orthogonal prime-editor scan of the same intended variants; restrict library to bystander-free spacers when possible; deconvolute via allele-frequency table. ### sgRNA shows perfect editing but no fitness signal **Trigger:** Intended variant is silent or compensatory; the protein function is unchanged. **Mechanism:** Variants can be tolerated; not all variants are LoF or GoF. **Symptom:** High editing efficiency (>70%) but per-sgRNA LFC near zero. **Fix:** Expected outcome for many variants; flag silent / compensatory variants in the report. ### Low editing across all guides **Trigger:** Wrong cell line for the BE; cell line has poor BE activity (some lines lack APOBEC or have low expression). **Mechanism:** BE efficiency depends on cell-line expression of TadA or APOBEC components. **Symptom:** Median editing <30% across library. **Fix:** Test in a BE-validated cell line (HEK293T, U2OS, K562 generally work); pilot before full screen. ### Library missing intended-variant sgRNAs **Trigger:** No NGG-adjacent spacer places target base in editing window for that codon. **Mechanism:** Editor window is fixed; some codons cannot be targeted with given chemistry. **Symptom:** Specific variants absent from screen. **Fix:** Use PAM-relaxed BE variants (SpRY-CBE, SpRY-ABE); use prime editor for variants outside BE accessibility; accept that some variants cannot be installed. ## Quantitative Thresholds | Threshold | Value | Source / Rationale | |-----------|-------|--------------------| | Editing window | Positions 4-8 from PAM-distal end (BE3/BE4); 4-10 (ABE8e) | Komor 2016; Gaudelli 2017; Lapinaite 2020 | | Editing efficiency for screen power | >30% (primary); >50% (validation) | Hanna 2021 conventions | | Indel byproduct (clean BE) | <5%; <2% for ABE | Koblan 2018 (BE4max); Gaudelli 2017 (ABE) | | Substitution-vs-indel ratio | >10 (clean BE); <3 (Cas9-like) | CRISPResso2 diagnostic | | Target editing % for variant inclusion | >30% Hanna 2021; >50% strict | Empirical | | Bystander rate (target attribution) | <10% acceptable; <5% ideal for clean attribution | Application-dependent | | Cell-line BE activity (pilot) | >30% editing at validated target | Below = wrong cell line for BE | | Per-amino-acid sgRNA density | 10-15 (Hanna 2021); 5-8 (smaller screens) | Tradeoff with library size | ## Common Errors | Error / symptom | Cause | Solution | |-----------------|-------|----------| | Substitution-vs-indel ratio <3 | Cas9 contamination or wrong BE | Verify vector / cell line; pilot first | | All edits at bystander positions | Target base outside window | Re-design spacer with target at pos 4-8 | | Variant attribution unclear | Bystander confounding | Run orthogonal PE; restrict library | | Library lacks intended variant | No NGG-PAM accessibility | SpRY-CBE; prime editor; accept exclusion | | Editing <30% library-wide | Cell-line BE inactivity | Re-validate cell line | | Hit list dominated by single sgRNA | Bystander-driven phenotype | Cross-check with bystander-free sgRNAs | ## References - Komor AC et al. 2016. *Nature* 533:420. BE3. - Gaudelli NM et al. 2017. *Nature* 551:464. ABE7.10. - Koblan LW et al. 2018. *Nat Biotechnol* 36:843. BE4max + improved CBE. - Lapinaite A et al. 2020. *Science* 369:566. ABE8e mechanism. - Richter MF et al. 2020. *Nat Biotechnol* 38:883. ABE8.20. - Sanson KR et al. 2020. *Nat Commun* 11:5165. GRACE library for BE screens. - Hanna RE et al. 2021. *Cell* 184:1066. Massively parallel BRCA1/2 variant function via CBE. - Cuella-Martin R et al. 2021. *Cell* 184:1081-1097. CBE saturation across 86 DDR genes (BRCA1/2 plus others). - Arbab M et al. 2020. *Cell* 182:463. BE-Hive prediction of editing outcomes. - Anzalone AV et al. 2019. *Nature* 576:149. Prime editing (PE2/PE3). - Clement K et al. 2019. *Nat Biotechnol* 37:224. CRISPResso2. - Kurt IC et al. 2021. *Nat Biotechnol* 39:41. CGBE1. ## Related Skills - crispr-screens/crispresso-editing - CRISPResso2 BE/PE mode and allele tables - crispr-screens/library-design - GRACE-style BE library design - crispr-screens/prime-editing-screens - Orthogonal PE for variant attribution - crispr-screens/hit-calling - Variant-level hit aggregation - crispr-screens/screen-qc - Editing-efficiency QC - crispr-screens/drugz-chemogenomic - drugZ for BE drug-modifier screens - clinical-databases/clinvar-lookup - Variant pathogenicity annotation - variant-calling/variant-annotation - VEP for predicted amino acid changes