TITLE: gRNA design and cloning with BbsI into Loop plasmid L1_lacZgRNA-Ck2/3
AUTHORS: Eftychis Frangedakis, Marta Tomaselli, Susana Sauret-Gueto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to design and clone the guide RNA target sequence into a L1 plasmid ready to accept the gRNA by cloning with BbsI. L1 plasmids are L1_lacZgRNA-Ck2 and L1_lacZgRNA-Ck3.</div><div class = "text-block"><span>If one gRNA target sequence is cloned into the Ck2 plasmid and another one into Ck3 one, the two L1_gRNA transcription units can be combined with an antibiotic resistance transcription unit and a MpEF1α:Cas9 transcription unit via </span><a href="https://www.protocols.io/view/loop-l2-even-level-sapi-type-iis-cloning-into-pcs-92fh8bn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">L2 SapI Loop assembly.</span></a><span> This allows for dual gRNA editing.</span></div></div>

[STEPS]
?. [Summary of design of gRNA and cloning into L1 with BbsI]
?. [Protocol for design of gRNA and cloning into L1 with BbsI]
gRNA oligo designOrder two oligos that contain the forward and reverse guide sequence plus the overhangs necessary for ligation (highlighted with bold) into L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3:oligo F: 5’-  CTCG-NNNNNNNNNNNNNNNNNNNN-gt 3’oligo R: 5’-  TAAAac-NNNNNNNNNNNNNNNNNNNN-3’Note: Standard de-salted oligos are ok
?. [Protocol for design of gRNA and cloning into L1 with BbsI]
Oligo annealingMix oligos with water as follow:oligo F (100μM)   1μloligo R (100μM) 1μl water  8μl Total volume  10μlAnneal in a thermocycler using the following parameters: 37oC for 30 min, 95oC for 5 min and then ramp down to 25oC at 5oC per min. After annealing the gRNA can be directly cloned into L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3 plasmid without the need of any further processing (step 4).
?. [Protocol for design of gRNA and cloning into L1 with BbsI]
Cloning into backbone vector In a 0.2 mL tube set up the following reaction:  AB1  Component
    Volume (μL)
  2Sterile water6310x T4 ligase buffer (NEB)141 mg/mL bovine serum albumin (NEB)0.55  T4 DNA ligase (5 U/µL) (Thermo Fisher)
  0.256BbsI (BpiI) (5 U/µL) (Thermo Fisher)  0.25 
  7L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3 (25-50 ng)18annealed oligo19  Final volume
  10 Place samples on the thermocycler and incubated using the following program: Assembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40 μg/mL for blue-white screening. Incubate at 37oC for 16 h. Confirm with sequencingAssembly:     15 cycles: 3 minutes at 37oC and 4 minutes at 16oC Termination: 5 minutes at 50oC and 10 minutes at 80oC  Transform chemically competent using 1 μL of reaction and plate on LB agar plates with 100 μg/mL spec and X-gal 40 μg/mL for blue-white screening. Incubate at 37oC for 16 h. Confirm with sequencing
AB1  Component
    Volume (μL)
  2Sterile water6310x T4 ligase buffer (NEB)141 mg/mL bovine serum albumin (NEB)0.55  T4 DNA ligase (5 U/µL) (Thermo Fisher)
  0.256BbsI (BpiI) (5 U/µL) (Thermo Fisher)  0.25 
  7L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3 (25-50 ng)18annealed oligo19  Final volume
  10