Table 1:

Summary of RAPID-DASH compared to other gRNA array approaches.

Study	Assembly approach	Maximum gRNAs assembled	gRNA processing method	Overall assembly time	Pre-cloning step	Scalability	Oligos pergRNA(length)	Other limitations
RAPID-DASH	Golden gate assembly	10 gRNAs(up to 20 gRNA units can beassembled usingRAPID-DASH online tool)	Individual expression cassettes	3 days	No	Scalable as gRNA oligos can be pooled during PCA toassemble gRNA arraylibraries	1 (59 bp)	Long read sequencing required to screen clones
Vad-Nielsen et al.26	Golden gate assembly	30 gRNAs(Assembled in 2 stages)	Individual expression cassettes	> 7 days	Yes	Cloning each gRNA restricts scalability	2 (25 bp)	Labor intensive:multiple rounds ofcloning, culturing and sequence validation
McCarty et al.18	Golden gate assembly	12 gRNAs	Csy4 processing	> 2 days	No	Scalable as the gRNA units can be pooledbefore adding Type IISoverhangs for the array assembly (Notdemonstrated in the study).	1 (53 bp)	Labour intensive: multiple rounds of PCR, digestion and gelpurifications. RequiresCsy4 endonuclease for individual gRNA processing.
Kurata et al.20	Golden gate assembly	10 gRNAs	Csy4 processing	> 5 days	Yes	Cloning each gRNA restricts scalability	2 (25 bp)	Labor intensive:multiple rounds ofcloning, culturing and sequence validation.Csy4 endonuclease for individual gRNA processing.
Zhang et al.23	Golden gate assembly	8 gRNAs	tRNA processing	>2 days	No	gRNA spacersequences being theGolden Gate overhangs restricts scalability.	2 (44 to 60 bp)	The gRNA maturation could interfere with endogenous tRNA pool
Yuan et al24	Golden gate assembly	10 gRNAs	tRNA/Csy4/Ribozyme processing	3–5 days	No	gRNA spacersequences being theGolden Gate overhangs restricts scalability.	2 (27 to 52 bp)	The gRNA maturation could interfere withendogenous tRNA pool(tRNA processing) or requires Csy4 endonuclease expression
Breunig et al.25	Gibson assembly	8 gRNAs	Individual expression cassettes	3 days	No	gRNA spacersequences mediatehomology for Gibson assembly thus restricting scalability.	2 (36–45 bp)	Low assembly efficiency(34% for 4 gRNA assembly)

^#Scalability indicates using the approach to assemble multiple gRNA arrays or libraries of arrays simultaneously.