Final answer:
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:
- Engineer gDNA with a Cas-specific DNA sequence that targets the desired sites.
- Fuse the gDNA to tracr DNA to create a single guide DNA (sgDNA) that can be transcribed into a single guide RNA (sgRNA).
- Engineer a CRISPR/Cas9 gene array that incorporates the sgDNA.
- Place the engineered array in a plasmid with regulated promoters.
- Use electroporation to transform the cells with the engineered array.
- 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.