✂️ CRISPR / Cas9

CRISPR gRNA Design Tools

Use dedicated external tools for guide RNA (gRNA) design and off-target evaluation. The following are external links to two widely used standard gRNA design tools.

CRISPick
portals.broadinstitute.org/gppx/crispick

The official sgRNA design tool from the Broad Institute's Genome Perturbation Platform. Simply enter a gene ID or RefSeq ID to design sgRNAs for CRISPRko, CRISPRa, and CRISPRi modes. Supported enzymes: SpyCas9 (NGG), SaurCas9 (NNGRR), AsCas12a (TTTV), enAsCas12a. Uses Azimuth 2.0 (Rule Set 2; Doench et al. 2016) for on-target scoring; off-target evaluated by CFD score. Supports large-scale library screening design. Successor to the GPP sgRNA Designer.

CRISPRko CRISPRa CRISPRi SpyCas9 SaurCas9 AsCas12a enAsCas12a Azimuth 2.0 CFD score Library Design
Open CRISPick →
CRISPOR
crispor.gi.ucsc.edu

An open-source gRNA design tool (also hosted on UCSC Genome Browser) where you can paste DNA sequences directly. Supports 700+ genomes and displays multiple on-target and off-target scores (MIT score, CFD score, Doench 2016, etc.) in one view. Automatically designs cloning oligos, PCR primers, and NGS validation primers. Compatible with SpCas9 (NGG), SaCas9, Cas12a, and many other enzymes. Especially useful for non-model organisms, custom genomes, and detailed off-target analysis.

Direct Sequence Input 700+ Genomes Multiple Score Comparison SpCas9 / SaCas9 Cas12a Support Cloning Sequences NGS Primers Open Source
Open CRISPOR →
📋 Tool Comparison
Feature CRISPick CRISPOR
Input Format Gene ID / RefSeq ID / Sequence DNA sequence (paste directly)
Cas Enzyme Support SpyCas9 (NGG), SaurCas9 (NNGRR), AsCas12a (TTTV), enAsCas12a SpCas9, SaCas9, Cas12a, and many more (custom PAM supported)
CRISPR Mode ✓ ko, a, i — all supported Knockout (cleavage) only
On-target Score Azimuth 2.0 (Rule Set 2) Rule Set 1, Doench 2016, multiple options
Off-target Analysis CFD score + Tier classification MIT score, CFD score, genome-wide search
Supported Genomes Human, Mouse, Rat 700+ species (custom genome supported)
Cloning Sequences ✓ Oligo pairs ✓ Oligos, PCR & NGS primers
Best Used When Knocking out or controlling expression (CRISPRa/i) of known genes
in human, mouse, or rat		 Working with any DNA sequence, non-model organisms,
or performing detailed off-target analysis
🧬 How CRISPR/Cas9 Works

Mechanism of CRISPR/Cas9 Genome Editing

CRISPR/Cas9 is a genome editing system in which guide RNA (gRNA) recognizes the target DNA sequence, and Cas9 nuclease cleaves the double strand. The design of the gRNA spacer sequence (20 nt) greatly affects both on-target efficiency and off-target risk.

Step 1
gRNA Design
Select 20 nt upstream of the PAM (NGG) from the target gene's exon sequence as the spacer sequence. Narrow down candidates based on 40–70% GC content, polyT avoidance, and high efficiency scores.
Step 2
Cas9 Cleavage
The gRNA hybridizes to the target protospacer, and Cas9 generates a double-strand break (DSB) 3 bp upstream of the PAM.
Step 3
Editing via DNA Repair
NHEJ (Non-Homologous End Joining) → Indel-mediated knockout.
HDR (Homology-Directed Repair) → Sequence knock-in using a donor template.
🖥 GeneQuick — Personal Gene Sequence Design & Editing App

A lightweight desktop app for researchers and students to use on their own PC.

Free Download →