Dec 04, 2019

Public workspacegRNA design and cloning with SapI into Loop plasmid L2_lacZgRNA-Cas9-CsA

 Forked from gRNA design and cloning into Loop L2 plasmids (L2_gRNA-Cas9-CsA and L2_gRNA-CsA plasmids)
DOI
dx.doi.org/10.17504/protocols.io.93wh8pe
  • 1University of Cambridge;
  • 2Plant Sciences, University of Cambridge, OpenPlant
  • OpenPlant Project
Susana Sauret-Gueto
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DOI: dx.doi.org/10.17504/protocols.io.93wh8pe
Protocol Citation: Eftychis Frangedakis, Marta Marta Tomaselli, Susana Sauret-Gueto 2019. gRNA design and cloning with SapI into Loop plasmid L2_lacZgRNA-Cas9-CsA. protocols.io https://dx.doi.org/10.17504/protocols.io.93wh8pe
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 03, 2019
Last Modified: December 04, 2019
Protocol Integer ID: 30550
Abstract
This protocol explains how to design and clone the guide RNA target sequence into a L2 plasmid ready to accept the gRNA by cloning with SapI (L2 plasmid also contains a cassette to express Cas9)

Summary of design of gRNA and cloning into L2 with SapI
Summary of design of gRNA and cloning into L2 with SapI
Design of oligos for gRNA SapI mediated cloning into L2_lacZgRNA-Cas9-CsA vector. The L2_lacZgRNA-Cas9-CsA SapI digested vector has AGC and TTT overhangs. Therefore, oligos for gRNA should be designed such that the forward strand has a 5' overhang of TCG and the reverse strand has a 5' overhang of gt-AAA (addition of “gt” nucleotides is necessary to reconstitute the full sequence of the gRNA scaffold in pink).Blue arrows: SapI recognition site. Blue dashed lines: SapI cleavage site. LacZ: lacZα cassette for blue-white screening of colonies.

Protocol for design of gRNA and cloning into L2 with SapI
Protocol for design of gRNA and cloning into L2 with SapI
gRNA oligo design

Order two oligos that contain the forward and reverse guide sequence plus the overhangs necessary for ligation (highlighted with bold) into L2_lacZgRNA-Cas9-CsA:

oligo F: 5’- TCG-NNNNNNNNNNNNNNNNNNNN-gt 3’
oligo R: 5’-AAAac-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 L2_lacZgRNACas9-CsA 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 5
10x Tango buffer (Thermo Fisher) 1
   1 mg/mL bovine serum albumin (NEB)    0.5
T4 DNA ligase (5 U/µL) (Thermo Fisher)   0.25
10mM ATP (SIGMA)     1
SapI (LguI) (5 U/µL) (Thermo Fisher) 0.25
L2_lacZgRNACas9-Csa (25-50 ng)1
annealed oligo1
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. Incubate at 37 oC for 16 h.

  • Confirm with sequencing