Question

In general, if there are multiple genes of interests, or edits at multiple points along a large gene are desired, can several edits done at once (or as many as is possible while minimizing off-target effects), transfecting cells with multiple gRNAs and possibly different Cas variants, or would the edits be done one at a time, with each edit being screened for one after another? Or somewhere in-between where you'd batch edits and screen for them in groups? Mostly interested in base edits and electroporation based transfection, but any information on workflow for editing multiple sites is welcome! Per bob1, my goal is to edit multiple base pairs on sites adjacent to and within a gene for in vitro mammalian cell lines, to see how those edits affect protein expression.

Answer 1

When using CRISPR/Cas for multiple gene edits, you can transfect cells with multiple gRNAs and different Cas variants or edit the genes one at a time and screen them individually. For base edits and electroporation-based transfection, you can follow a workflow that involves engineering gDNA, creating sgRNA, engineering a CRISPR/Cas9 gene array, transforming cells through electroporation, and activating the promoter to initiate editing.

Step-by-step explanation:

Multiple Gene Edits with CRISPR/Cas

When it comes to editing multiple genes or making multiple edits along a large gene using CRISPR/Cas, there are a few approaches. One option is to transfect cells with multiple gRNAs and possibly different Cas variants to achieve several edits at once. This method can help minimize off-target effects and increase efficiency. Another approach is to batch the edits and screen them in groups, where each edit is done one at a time and screened for individually. This allows for a more controlled and systematic editing process.

For base edits and electroporation-based transfection, you can follow these general steps:

1. Engineer gDNA with a Cas-specific DNA sequence that targets the desired sites.
2. Fuse the gDNA to tracr DNA to create a single guide DNA (sgDNA) that can be transcribed into a single guide RNA (sgRNA).
3. Engineer a CRISPR/Cas9 gene array that incorporates the sgDNA.
4. Place the engineered array in a plasmid with regulated promoters.
5. Use electroporation to transform the cells with the engineered array.
6. Activate the promoter to transcribe the CRISPR/Cas9 genes and initiate the editing process.

These steps can be modified based on your specific experimental design and requirements.