CRISPR gRNA Design for Diverse Applications

PE uses a nuclease-reverse transcriptase fusion protein and a unique prime editing gRNA (pegRNA) to introduce a range of edit types, including knock-ins and single-base changes. If you’d like to learn more about PE, we recommend this article as a starting point.

PE is unique among CRISPR editing methods because the gRNA determines the edit site and encodes the desired repair. Given the complexity of the pegRNA, we recommend using a specialized design tool like PE-Designer or the Prime Editing Design Tool to simplify your design process.

When designing and optimizing pegRNAs for your experiment, we recommend considering the following parameters (Anzalone et al.).

• Primer binding site (PBS). The PBS portion of your pegRNA binds the non-target strand and primes the reverse transcriptase-mediated repair that will introduce the edit encoded by your pegRNA.
  • Length. A PBS length of approximately 13 nucleotides is generally optimal, though we recommend testing alternative lengths if the GC content of the PBS is outside a 40-60% range.
  • Sequence. If feasible, select a PBS sequence with a 40-60% GC content. Priming regions with GC content below ~40% may require longer PBS sequences.
• Reverse transcriptase template (RTT). The RTT portion of your pegRNA encodes the desired edit.
  • Length. The ideal RTT length is target site-dependent. An RTT length of 10-16 nucleotides is optimal for most sites, though we recommend testing longer and shorter RTT sequences as needed. For larger insertions, your RTT length may fall well outside this range. In these cases, we recommend testing a range of RTT lengths and sequences to identify the best RTT for your specific application.
  • Sequence. Avoid selecting an RTT with a C in the 5’-most position. pegRNAs with a C in this position tend to drive lower editing efficiencies, likely due to their impact on gRNA structure.
• Target sequence. The target sequence of your pegRNA guides the PE complex to the appropriate genomic site. As for any CRISPR experiment, please confirm the specificity of your target sequence and ensure that your selected gRNA positions the prime editor’s nick locations appropriately relative to your intended edit.

CRISPRa and CRISPRi are non-editing applications of CRISPR technology. Instead, a catalytically inactive Cas nuclease, or dead Cas (dCas), is fused to a transcriptional effector that promotes (CRISPRa) or represses (CRISPRi) transcription. If you’d like to learn more about CRISPRa/CRISPRi, we recommend our comprehensive guide to CRISPR methods and this introduction to CRISPRa/i.

For CRISPRa and CRISPRi experiments, you must design gRNAs that target the promoter region of your gene of interest. This type of design can be facilitated by a specialized design tool, like CRISPR-ERA or CRISPick, which suggests designs based on the annotated start site of your gene. Other design tools, like Benchling, can also work well for CRISPRa/CRISPRi gRNA design, though you may need to manually identify the target region (promoter).

Introducing two or more gRNAs into your target cells simultaneously can result in the excision (deletion) of the genomic fragment between the cut sites.

In CRISPR experiments involving two or more gRNAs, the frequency of specific fragment deletions depends on a number of factors, including gRNA spacing. Cut sites separated by larger distances are more likely to yield indels at each cut site versus the deletion of the sequence between the cut sites. While fragment deletions of >1 kb in size are possible, a 2020 study demonstrated that gRNA spacing of 45-300 bp was most likely to result in synergistic editing. For smaller fragment deletions, we advise selecting gRNAs that do not overlap because they may compete with one another.

Most CRISPR gRNA design tools can be used to identify gRNAs for fragment deletion edits. For this edit type, we recommend using a tool like Benchling that allows you to visualize the relative position of each gRNA within the genomic region you plan to target.

Once you are ready to order your gRNAs, you can access our ordering page here.

For a comprehensive guide to Synthego’s CRISPR resources, please visit our Getting Started with CRISPR page.