Table 3. Comparison of genome editing techniques.

	No-SCAR	TetA-SacB Dual Selection5	SceI counter-selection10	Datsenko and Wanner1
Day 1	1) Transform pCas9cr4 2) Clone spacer (2 fragments)1,3	Transform λ-Red plasmid	1) Transform λ-Red plasmid 2) Clone Mutation Cassette (4 fragments)2,3	1) PCR donor DNA 2) Transform λ-Red plasmid pKD46 into target strain
Day 2	Grow clones	Grow cells overnight	Screen and sequence clones	Transform Linear DNA
Day 3	Isolate plasmid and transform into cells with pCas9cr4	1) Subculture and induce λ-Red 2) Transform TetA-SacB Cassette	PCR amplify Mutation Cassette and Transform	Screen AbR colonies Cure λ-Red
Day 4	1) Start culture and induce λ-Red 2) Transform linear DNA, induce Cas9	Restreak colonies on counter-selection medium	Restreak Colonies	Transform pCP20
Day 5	1) Screen colonies 2) Grow at 37 to cure pKDsgRNA4	Identify sucroses clones and start overnight culture	Resuspend colonies and plate to induce DSB	Express Flp recombinase
Day 6	Transform pKD-p15, induce Cas9	1) Start Culture and induce λ-Red 2) Transform with linear DNA and plate on counterselection medium	Patch colonies to screen for AbS	PCR screen or Patch for AbS Grow at 37 to cure plasmid
Day 7	Patch colonies for cms, Grow at 37°	Incubate at 42°	Passage cells to cure plasmid4	Plasmid free colonies
Day 8	Plasmid free colonies	Screen by PCR or TetS	Plasmid free colonies	-
1 mutation	5 Days	8 Days	7 Days	6 Days
1 mutation w/ curing	8 Days	8 Days	8 Days	7 Days
2 mutations w/ curing	113 Days	16 Days	134 Days	13 Days
3 mutations w/ curing	14 Days	24 Days	18 Days	19 Days
Essential Genes	No additional requirements	Must express in-trans	Special considerations	N/A

¹Requires 2 fragment ligation independent cloning, with the primer designs given in the manuscript we have been 100% successful in producing these clones.

²Requires 4 fragment ligation independent cloning that should be sequence verified before transformation.

³When multiple mutations are desired cloning can be performed simultaneously on day 1, resulting in a faster turnaround time for subsequent mutations.

⁴For more than one mutation the strains obtained during day 5 can be cured of plasmid and immediately transformed with new pKDsgRNA, resulting in 3 day turnaround for each additional mutation.

⁵For more than one mutation the strain obtained on day 7 can be transformed with Linear DNA (Day 3) resulting in a 5 day turnaround for each additional mutation.