Q: How do I handle bystander edits in base editing screens?

Partition CRISPResso2 allele tables by exact edit pattern to separate target-only from target-plus-bystander reads. Tile multiple sgRNAs with different bystander patterns per variant, or cross-validate with prime editing, which produces no bystanders.

Q: What editing efficiency threshold should I use before hit calling?

Drop sgRNAs below 30% target-base conversion for primary screening and require above 50% for validation-grade hits. Unedited reads carry no biological perturbation, so including low-efficiency guides dilutes signal and adds noise.

Q: Why does my base editing sample show mostly indels?

A substitution-vs-indel ratio below 3 indicates Cas9 contamination or a wrong vector rather than clean base editor activity. Verify the construct uses nCas9-BE rather than wild-type Cas9 and confirm the cell line lacks Cas9 background expression.

Q: When should I use prime editing instead of base editing?

Use prime editing when the target base falls outside editing positions 4-8, when zero bystanders are required, or for transversions and multi-base edits. Base editors are preferred for C-to-T or A-to-G transitions due to higher efficiency.

Question 1

How do I analyze a base editing screen with CRISPResso2?

Accepted Answer

Run CRISPResso2 with --base_editor_output and the appropriate --conversion_nuc_from/to flags to quantify per-position base conversion. Then filter sgRNAs by editing efficiency, call hits with MAGeCK MLE or drugZ, and aggregate sgRNA scores to per-variant fitness.

Question 2

CBE vs ABE: which base editor should I use for my variant screen?

Accepted Answer

Use CBE (BE4max) for C-to-T edits and ABE (ABE7.10 or ABE8e) for A-to-G edits. ABE7.10 has the narrowest window (positions 4-7) and lowest indel rate (<2%), making it cleanest when bystander edits must be minimized.

Question 3

How do I handle bystander edits in base editing screens?

Accepted Answer

Partition CRISPResso2 allele tables by exact edit pattern to separate target-only from target-plus-bystander reads. Tile multiple sgRNAs with different bystander patterns per variant, or cross-validate with prime editing, which produces no bystanders.

Question 4

What editing efficiency threshold should I use before hit calling?

Accepted Answer

Drop sgRNAs below 30% target-base conversion for primary screening and require above 50% for validation-grade hits. Unedited reads carry no biological perturbation, so including low-efficiency guides dilutes signal and adds noise.

Question 5

Why does my base editing sample show mostly indels?

Accepted Answer

A substitution-vs-indel ratio below 3 indicates Cas9 contamination or a wrong vector rather than clean base editor activity. Verify the construct uses nCas9-BE rather than wild-type Cas9 and confirm the cell line lacks Cas9 background expression.

Question 6

When should I use prime editing instead of base editing?

Accepted Answer

Use prime editing when the target base falls outside editing positions 4-8, when zero bystanders are required, or for transversions and multi-base edits. Base editors are preferred for C-to-T or A-to-G transitions due to higher efficiency.

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