sgRNA Design Rules for CRISPR

Overview

• Topic: Designing single guide RNA (sgRNA) sequences for CRISPR/Cas9 gene editing.
• Focus: rules for selecting target sequences, design tips, production and screening of sgRNAs.
• Source: Educational content describing practical guidelines and recommended kits.

Key Concepts

• sgRNA components:
  • crRNA: 20-nt targeting sequence homologous to genomic DNA.
  • tracrRNA: Cas9-recruiting scaffold sequence.
• PAM requirement:
  • Cas9 requires a proto-spacer adjacent motif (PAM) 5'-NGG-3' immediately downstream (3' end) of target.
  • PAM is required for cleavage but is not included in the sgRNA sequence.
• Cleavage location:
  • Cas9 typically cleaves ~3 bases upstream of the PAM.

Choosing a Target Sequence

• Select 20 nt immediately upstream (5') of a 5'-NGG-3' PAM.
• Target can be on either DNA strand.
• Do not include the PAM in the sgRNA sequence.
• Use sgRNA design tools to:
  • Detect PAMs and list candidate 20-nt crRNA sequences.
  • Predict potential off-target sites across the genome.
• Recommended practice: design multiple sgRNAs per gene because activity and specificity are unpredictable.

Design Tips (empirical)

• Preferred nucleotides observed in effective sgRNAs:
  • Position 1 (5' end of crRNA): G (guanine) is often beneficial.
  • Position 17: A or T may improve performance.
• These are empirical observations and not absolute rules.

Producing sgRNAs

• Common method: in vitro transcription from a PCR-generated template.
  • Template contains T7 promoter, crRNA sequence and tracrRNA sequence.
  • Purify the transcribed sgRNA for testing or cell delivery.
• Example product mentioned: Guide-it sgRNA In Vitro Transcription Kit (uses PCR to generate T7-driven template and then in vitro transcription).

Screening and Using sgRNAs

• Because sgRNA efficiency is variable, screen candidate sgRNAs before cell transduction.
• In vitro screening:
  • Use an sgRNA screening assay to identify the most efficient guides.
  • Example product: Guide-it sgRNA Screening Kit for in vitro efficiency assessment.
• After selecting the most effective sgRNA(s), proceed to transduction or delivery in cells.

Key Terms and Definitions

• sgRNA: Single guide RNA combining crRNA and tracrRNA sequences for Cas9 targeting.
• crRNA: Guide portion (20 nt) complementary to genomic target.
• tracrRNA: Structural portion that recruits Cas9.
• PAM (proto-spacer adjacent motif): Short DNA motif (5'-NGG-3') required for Cas9 binding and cleavage.
• In vitro transcription: Enzymatic synthesis of RNA from a DNA template (often driven by T7 polymerase).

Practical Workflow Summary (table)

Action Items / Next Steps (if applying these guidelines)

• Use an sgRNA design tool to generate candidate 20-nt guides near NGG PAMs.
• Design at least 2–4 different sgRNAs per target gene to increase success chance.
• Order or prepare PCR templates for in vitro transcription (include T7 promoter).
• Perform in vitro transcription and purify sgRNAs.
• Screen sgRNAs in vitro to select top performers before cell experiments.
• Validate on-target editing and assess off-target effects after cell editing.

References Mentioned (for further study)

• Online sgRNA design tools page (collection of resources for guide selection and off-target prediction).
• Guide-it In Vitro Transcription Kit (for sgRNA production).
• Guide-it sgRNA Screening Kit (for in vitro assessment of guide efficiency).

Notes / Limitations

• Guidelines reflect empirical observations and standard Cas9 (SpCas9) PAM requirements (NGG).
• Always confirm compatibility with the specific Cas9 variant or delivery system used.
• No experimental protocols or parameter details (concentrations, times) are included here; follow kit manuals or validated protocols.