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sgRNA Template Oligo Designer

Design DNA oligos for in-vitro transcription of CRISPR sgRNAs. Enter a 20-nt protospacer target, choose your Cas variant (SpCas9, SpCas9-NG, SaCas9), and get forward/reverse oligos with T7 promoter, annealing protocol, and IDT order sheet. All computation runs client-side.

CRISPR / sgRNASpCas9 / SaCas9Client-Side

Try it out

Load example sgRNA Template Oligo Designer data to see the full workflow

Target Protospacer Sequence

Enter your 20-nt protospacer target (ACGT only, no PAM). Whitespace and digits are stripped automatically.

Design Settings

PAM: 5′-NGG-3′ (downstream of target)

Required for T7 in-vitro transcription

  • Generate DNA oligos for T7 in-vitro transcription of sgRNAs
  • Design guides for SpCas9, SpCas9-NG, or SaCas9 CRISPR systems
  • Create IDT-compatible order sheets for rapid oligo ordering
  • Get a ready-to-use annealing protocol for sgRNA template preparation

Don't use for

  • Finding CRISPR target sites in a genome — use CRISPOR or Cas-OFFinder first, then bring the 20-nt target here
  • Designing cloning-based sgRNA expression constructs (e.g., lentiGuide vectors) — those use BsmBI golden gate cloning, not IVT
  • Base editing or prime editing guide design — those have additional sequence requirements beyond the protospacer

How CRISPR-Cas9 genome editing works

CRISPR-Cas9 is a genome editing system derived from bacterial adaptive immunity. The system has two components:

1. Guide RNA (sgRNA): A ~100-nt RNA that contains a 20-nt protospacer complementary to the target DNA, fused to a scaffold sequence that binds the Cas9 protein.
2. Cas9 protein: An endonuclease that creates a double-strand break (DSB) at the target site. Cas9 requires a PAM (protospacer adjacent motif) immediately downstream of the target for recognition.

After Cas9 creates a DSB, cellular repair pathways (NHEJ or HDR) introduce insertions, deletions, or precise edits at the target locus.

In-vitro transcription workflow for sgRNA

To produce sgRNA by in-vitro transcription (IVT):

1. Design oligos: This tool generates the forward (T7 + protospacer + scaffold overlap) and reverse (scaffold complement) oligos 2. Anneal oligos: Heat to 95 °C, slow-cool to 25 °C to form a partially dsDNA template 3. IVT reaction: T7 RNA polymerase transcribes from the dsDNA promoter region, using the single-stranded scaffold overhang as a template 4. Purify: Column or phenol-chloroform purify the sgRNA, DNase-treat to remove template 5. Deliver: Electroporate or transfect sgRNA + Cas9 protein (RNP complex) into cells

Choosing a good target site

Tips for selecting an effective CRISPR target:

  • GC content: Aim for 40–70% GC. Very low or high GC reduces sgRNA activity
  • Avoid homopolymers: Runs of 4+ identical bases (especially TTTT, which acts as a Pol III terminator) can reduce expression
  • Start with G: If using T7 IVT, a leading G improves transcription efficiency
  • Check off-targets: Use Cas-OFFinder or CRISPOR to verify specificity before ordering oligos
  • Position in gene: For knockouts, target early exons (but not exon 1, which may have alternative start sites)
  • Avoid SNPs: Common variants in the target or PAM reduce cutting efficiency in some genotypes

Frequently Asked Questions

Next Steps

PCR Fidelity Estimator

Estimate mutation rates for genotyping PCR after CRISPR editing

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DNA / RNA Bio Converter

Convert between mass, moles, and copies for sgRNA quantification

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Gibson Assembly Reaction Calculator

Calculate Gibson assembly reactions for constructing donor templates

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Electroporation Systems

Deliver sgRNA-Cas9 RNP complexes into cells by electroporation

Browse →

Thermal Cyclers

Anneal oligos and run downstream genotyping PCR

Browse →

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