How are the homology arms for Alt-R™ HDR Donor Oligos and Alt-R HDR Donor Blocks selected by the HDR Design Tool?
For wild-type Cas9, one of the homology arms is positioned relative to the CRISPR cut site, and the other is positioned relative to the specific mutation site. The default homology arm length for standard single-stranded Alt-R™ HDR Donor oligo designs is 40 bases. The default homology arm length for longer double-stranded Alt-R HDR Donor Block designs is 200 bp. Note that we recommend using homology arms of 200-300 bp. However, homology arms as short as 100 bp can be used if needed due to sequence complexities in the longer homology arm regions.
For nickase designs using single-stranded Alt-R HDR Donor oligos, there are two CRISPR nick sites rather than a single cut site. The mutation is typically located between the two guides, and the homology arms are positioned relative to these two CRISPR nick sites