For point mutation, it is suggested to use asymmetric ssDNA design. You can read this paper for more design tips. For large gene insertion, homology arms with 300-1000 bp flanking the insertion gene have been reported. In most cases, 500 bp long homology arms should work. It is important to design the ssDNA template containing a silent mutation to mutate the sgRNA PAM sequence in order to avoid secondary cleavage. KI donor design can be complicated. It is highly suggested to use a software (e.g. snapgene) to view and edit the sequence.
Articles in this section
- How to choose from various HDR templates when performing CRISPR mediated gene KI? Should I choose dsDNA, plasmid DNA, or ssDNA?
- What are the tips for designing an effective ssDNA template?
- What is the recommended homology arm length on each side of the template DNA when designing ssDNA?
- Any tips for improving CRISPR KI efficiency?
- Any tips for generation of conditional alleles in mouse?
- What if low HDR efficiency is detected in transfected cells?
- What is the maximum ssDNA length GenScript produces?
- What is the maximum quantity GenScript provides?
- What are the quality control tests you provide for the ssDNA templates?
- Why do you do double-sequencing, including the final ssDNA product?