Dec 15, 2025
gRNA design and cloning with BbsI into Loop plasmid L1_lacZgRNA-Ck2/3
- Eftychis Frangedakis1,
- Marta Tomaselli2,
- Susana Sauret-Gueto2
- 1University of Cambridge;
- 2Plant Sciences, University of Cambridge, OpenPlant
- Hornworts
Protocol Citation: Eftychis Frangedakis, Marta Tomaselli, Susana Sauret-Gueto 2025. gRNA design and cloning with BbsI into Loop plasmid L1_lacZgRNA-Ck2/3. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp1e8jgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 15, 2025
Last Modified: December 15, 2025
Protocol Integer ID: 235037
Keywords: l1-grna transcription unit, cas9 transcription unit via l2 sapi loop assembly, grna target sequence, guide rna target sequence, plasmid l1-laczgrna, cas9 transcription unit, antibiotic resistance transcription unit, dual grna, grna design, grna, rna, l2 sapi loop assembly, bbsi into loop, mpef1α, cas9
Abstract
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.
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 L2 SapI Loop assembly. This allows for dual gRNA editing.
Troubleshooting
Summary of design of gRNA and cloning into L1 with BbsI
Design of oligos for gRNA BbsI mediated cloning into L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3 vectors. The L1_lacZgRNA-Ck2/3 BbsI digested vectors have GAGC and TTTA overhangs. Therefore, oligos for gRNA should be designed such that the forward strand has a 5' overhang of CTCG and the reverse strand has a 5' overhang of CA-AAAT (addition of “CA/GT” nucleotides is necessary to reconstitute the full sequence of the gRNA scaffold in pink). Light brown arrows: BbsI recognition site. Light brown dashed lines: BbsI cleavage site. LacZ: lacZα cassette for blue-white screening of colonies.
Protocol for design of gRNA and cloning into L1 with BbsI
gRNA oligo design
Order 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
Oligo annealing
Mix oligos with water as follow:
oligo F (100μM) 1μl
oligo R (100μM) 1μl
water 8μl
Total volume 10μl
Anneal 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).
Cloning into backbone vector
In a 0.2 mL tube set up the following reaction:
| Component | Volume (μL) | |
| Sterile water | 6 | |
| 10x T4 ligase buffer (NEB) | 1 | |
| 1 mg/mL bovine serum albumin (NEB) | 0.5 | |
| T4 DNA ligase (5 U/µL) (Thermo Fisher) | 0.25 | |
| BbsI (BpiI) (5 U/µL) (Thermo Fisher) | 0.25 | |
| L1_lacZgRNA-Ck2 or L1_lacZgRNA-Ck3 (25-50 ng) | 1 | |
| annealed oligo | 1 | |
| 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 50μg/mL Kan and X-gal 40 μg/mL for blue-white screening. Incubate at 37oC for 16 h.
- Confirm with sequencing