What problem does it solve? Designing a pooled CRISPR screen library requires choosing the right chemistry, scoring guides for on-target activity and off-target liability, positioning them correctly relative to exons or transcription start sites, adding proper controls, and validating the cloned pool—mistakes in any step silently ruin the screen. ## Core Features & Use Cases - Chemistry and library selection: Decision tables for Cas9 KO, CRISPRi, CRISPRa, Cas12a multiplex, base-editor, and prime-editor screens, mapped to canonical libraries (Brunello, TKOv3, Dolcetto, Calabrese, Inzolia). - Guide scoring and selection: On-target scoring (Rule Set 2, DeepSpCas9, CRISPRon), off-target scoring (CFD, MIT) via CRISPOR, plus Python functions for candidate enumeration, exon-position filtering, and independent-guide selection. - TSS-relative positioning: CRISPRi (-50 to +300) and CRISPRa (-150 to -75) windows anchored to FANTOM5 CAGE peaks, with fallback handling for poorly annotated TSSs. - Controls, oligo design, and QC: Control-guide composition (~1% NTCs, safe-harbor, CEGv2/NEGv1), BsmBI cloning oligo construction, and plasmid-pool QC thresholds (Gini <0.1, skew <5, ≥99% guide detection). - Use Case: Ask the agent to design a Brunello-style Cas9 knockout library for 250 kinases with 4 guides per gene and 500 non-targeting controls, and receive a ranked guide table, oligo synthesis order, and coverage calculations. ## Quick Start Ask the agent to design a focused Cas9 knockout library for your gene list with 4 guides per gene, CFD off-target filtering, and standard non-targeting and essential-gene controls.
