Auto-SMART: a web-based platform for repair-template design for CRISPR/Cas9 homology-directed editing

CRISPR/Cas9 genome engineering has revolutionized biological research and gene therapy development, but it is limited by the positioning and quality of guide RNAs relative to the desired edit site. The SMART technique overcomes this constraint by subtly altering the gap between the Cas9 cut site and the edit in the repair template, preserving protein function while expanding design possibilities.

However, creating these templates manually requires complex coordination of multiple factors. Auto-SMART is a web-based platform that streamlines this process for CRISPR/Cas9-mediated insertions, deletions, point mutations, and sequence replacements in both coding exons and introns. Users specify the genome assembly, search for a gene or transcript, select a modification site, and choose an editing operation.

The platform identifies nearby Streptococcus pyogenes Cas9 (SpCas9) guides, ranks them by predicted on-target activity and cut-to-edit geometry, constructs repair templates with synonymous substitutions, automatically avoids re-cutting on residual on-target sites, and generates interpretable visual panels. These include the target locus, repair template, locus after HDR, and a downloadable report with a Primer3 genotyping handoff. The Auto-SMART web application is freely accessible at https://auto-smart.chuanping.org/.