/sgrna-design-guide

name: sgrna-design-guide
description: Three-tiered sgRNA design guide using validated Addgene sequences, CRISPick pre-computed datasets, or de novo design rules for CRISPR experiments
license: open

sgRNA Design Guide: Three-Tiered Approach

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Metadata

**Short Description**: Comprehensive guide for finding or designing sgRNAs using validated sequences, CRISPick datasets, or de novo design tools.

**Authors**: Ohagent Team

**Version**: 1.0

**Last Updated**: November 2025

**License**: CC BY 4.0

**Commercial Use**: Allowed

Citations and Acknowledgments

If you use validated sgRNAs from our database (Option 1):

• **Database Source**: Addgene (https://www.addgene.org)
• **Citation**: Always cite the original publication associated with each sgRNA using the PubMed ID provided in the database
• **Acknowledgment**: "Validated sgRNA sequences obtained from Addgene (https://www.addgene.org/crispr/reference/grna-sequence/)"

If you use CRISPick designs (Option 2):

• **Acknowledgment Statement**: "Guide designs provided by the CRISPick web tool of the GPP at the Broad Institute"
• **Citation for Cas9 designs (SpCas9, SaCas9)**: Sanson KR, et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nat Commun. 2018;9(1):5416. PMID: 30575746
• **Citation for Cas12a designs (AsCas12a, enAsCas12a)**: DeWeirdt PC, et al. Optimization of AsCas12a for combinatorial genetic screens in human cells. Nat Biotechnol. 2021;39(1):94-104. PMID: 32661438
• Note: This paper describes enAsCas12a optimization; specify which variant you used in your methods

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Overview

This guide provides a three-tiered approach to sgRNA design, prioritizing validated sequences before moving to computational predictions. Always start with Option 1 and proceed to subsequent options only if needed.

Key Concepts

Validated sgRNA Databases (Tier 1)

Validated sgRNAs are guide sequences that have been experimentally tested in published work, with documented cell line, cutting efficiency, and (often) off-target characterization. Addgene curates such sequences alongside the deposited plasmids that carry them, and a downloadable CSV (`addgene_grna_sequences.csv`) provides a searchable index keyed by gene symbol, target species, and application (cut / activate / RNA targeting). Using a validated sgRNA is preferred because the largest source of CRISPR experimental failure is poor on-target activity that would have been detected during the original validation.

Computational sgRNA Scoring (Tier 2)

When no validated sgRNA exists, pre-computed genome-scale designs from the Broad Institute's CRISPick service are the next best option. CRISPick provides 238 datasets covering multiple genomes, Cas variants, and applications, with each candidate guide ranked by **on-target efficiency** (how reliably it cuts), **off-target specificity** (how unlikely it is to cut elsewhere), and a **combined rank** that balances both. The on-target models behind CRISPick are Sanson 2018 (Cas9) and DeWeirdt 2021 (Cas12a). Combined Rank is the recommended default; use On-Target or Off-Target rank only when the experiment specifically prioritizes one over the other.

Off-Target Stringency and PAM Compatibility

PAM (Protospacer Adjacent Motif) requirements differ by Cas variant: SpCas9 needs NGG (3'), SaCas9 needs NNGRRT (3'), AsCas12a and enAsCas12a need TTTV (5'). Critically, **AsCas12a and enAsCas12a are different enzymes**: enAsCas12a is an engineered variant with broadened activity, and guides optimized for one will not perform identically on the other. Always match the CRISPick dataset to the exact Cas variant used in the lab. Off-target stringency thresholds (typically `Off-Target Rank` or a CFD-style score) trade specificity against the size of the candidate pool — tighter thresholds yield fewer but cleaner guides.

De Novo Design Rules (Tier 3)

When neither validated sequences nor pre-computed datasets cover the target (non-model organism, custom locus, etc.), apply rule-based design: 20 bp protospacer for SpCas9/SaCas9 (23–25 bp for Cas12a), GC content 40–60%, avoid TTTT (Pol III terminator) and homopolymer runs >4. Target location matters: early exons (first 50% of coding sequence) for knockout, −200 to +1 from TSS for CRISPRa, −50 to +300 from TSS for CRISPRi.

Decision Framework

sgRNA design decision tree
└── Does Addgene database (Tier 1) contain a validated sgRNA for your gene + species + application?
    ├── Yes -> Use the validated sgRNA(s); cite the original PubMed reference
    └── No  -> Run advanced literature search (mandatory before Tier 2)
        ├── Found in literature -> Use the literature sgRNA; cite the paper
        └── Still no match -> Is the target organism + Cas variant covered by a CRISPick dataset (Tier 2)?
            ├── Yes -> Download dataset, filter by gene, sort by Combined Rank
            │         └── Pick top 3-4 sgRNAs (ideally from different exons for redundancy)
            └── No  -> De novo design (Tier 3) using 20 bp / PAM / GC / avoid-TTTT rules
                      └── Validate experimentally (Sanger / T7E1 / amplicon-seq)

| Situation | Recommended tier | Rationale | |-----------|------------------|-----------| | Common human/mouse gene with prior CRISPR publications | Tier 1 (Addgene + literature) | Validated sequences come with measured cutting efficiency; lowest experimental risk | | Genome-scale screen of a model organism | Tier 2 (CRISPick) | Pre-computed datasets cover whole genomes with consistent scoring | | Single human gene knockout, no Addgene hit | Tier 2 (CRISPick GRCh38 SpCas9 CRISPRko), filter by Combined Rank | Best balance of efficiency and specificity for one-off knockouts | | CRISPRa / CRISPRi experiment | Tier 2 with the matching `CRISPRa` / `CRISPRi` dataset | Activation/inhibition models target proximal-promoter windows, not coding exons | | Cas12a (AsCas12a vs enAsCas12a) | Tier 2 with the **exact