Want to try RAPID-DASH for your own gRNA array assembly? We built a Shiny app to design the exact oligos you need — it automatically formats overhangs for Golden Gate cloning. Check it out here: deboerlab.shinyapps.io/OligoDesigner/ (10/11)

RAPID-DASH is also highly scalable. We used a pool of 10 gRNAs to assemble arrays with 10 units, resulting in randomized combinations of guides. The approach is easily extended to larger pools, making it well-suited for CRISPR screening and high-complexity library construction. (9/11)

But are the gRNAs actually functional in cells? Yes! We used a mutant GFP reporter line where GFP can only be restored by successful C→T base editing. All gRNAs in the array activated GFP—regardless of their position—showing robust expression and activity across the full array. (8/11)

But how efficient is the overall method? We went a step further... We sequenced bulk plasmid DNA (without picking individual colonies). The result? ~81% of arrays were perfectly assembled. Huge shoutout to @plasmidsaurus.bsky.social — this basically parallelizes the colony screening process. (7/11)

To confirm the assembly of full-length gRNA arrays, we did whole plasmid sequencing on the transformed clones. Every gRNA, in the correct order ✅. The consensus sequence from each clone matched the intended design exactly. (6/11)

How it works: Each gRNA unit is built via Polymerase Cycling Assembly (PCA) using a dsDNA U6 promoter, ssDNA 59nt spacer oligo (synthesized) and dsDNA scaffold terminator. PCR primers add Type IIS overhangs for ordered multi-fragment Golden Gate assembly. (4/11)

Introducing RAPID-DASH: Rapid Assembly of PCA-produced Individual DNAs and Directed Assembly through typeIIS overHangs. With RAPID-DASH, we assembled 10 gRNAs into an array using a 1-step cloning method with a single day of hands-on work. (3/11)