Custom Cas gRNA Library Designer: A Practical Guide to CRISPR Library Design

Designing a CRISPR gRNA library can become challenging when working with a large number of genes or with novel Cas proteins that recognize non-standard PAM sequences.

The Custom Cas gRNA Library Designer is a web-based pipeline that simplifies this process by supporting both whole-genome and pathway-specific gRNA library design. It also allows users to work with both already validated Cas proteins and novel or custom Cas variants with user-defined PAMs.

You can access the pipeline for free here:

Custom Cas gRNA Library Designer

1. Whole-Genome gRNA Library

If you want to design a gRNA library targeting genes across the genome, select the Whole Genome option.

You can then choose between:

• Already Validated Cas — for Cas proteins with predefined targeting and PAM requirements.
• Custom Cas — for novel or engineered Cas variants where you can define the relevant PAM and guide-design parameters.

This makes the pipeline useful for designing genome-wide libraries not only for established nucleases, but also for new Cas variants with novel PAM preferences.

The workflow is:

Whole Genome → Select Cas → Define PAM/parameters → Identify target sites → Generate gRNA library

2. Pathway-Specific gRNA Library

If you are interested in a particular biological process rather than the entire genome, select Pathway Specific.

The pipeline can retrieve genes associated with the selected pathway and subsequently identify compatible gRNA target sites.

For example, you could generate a focused library targeting genes involved in DNA repair, immune signaling, metabolism, or other biological pathways.

As with the whole-genome workflow, you can use either an already validated Cas or a custom Cas with a novel PAM.

The workflow is:

Pathway → Gene set → Select Cas → Identify target sites → Generate gRNA library

3. Which Option Should You Choose?

The pipeline essentially gives you four possible workflows:

This flexibility allows the same platform to be used for both routine CRISPR library design and emerging Cas-nuclease development.

Conclusion

The Custom Cas gRNA Library Designer provides a simple workflow for going from your genome or biological pathway and Cas nuclease to a candidate gRNA library.

Whether you are using an established Cas protein or developing a novel Cas variant with a new PAM, the pipeline can help streamline the guide-design process.

🎥 Want to see how the pipeline works in practice?

I have also prepared a step-by-step video tutorial showing how to use the different options and generate your gRNA library. Check out the video tutorial for a complete walkthrough of the pipeline. 

Open the Custom Cas gRNA Library Designer

This pipeline was developed as part of work supported by COST Action CA21113 (GeneHumDi).