Jun 12, 2024
Single-step assembly of double guide plasmid (pCas9-Duo) for gene-editing in Plasmodium
- 1Francis Crick Institute
Protocol Citation: Abhinay Ramaprasad 2024. Single-step assembly of double guide plasmid (pCas9-Duo) for gene-editing in Plasmodium . protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn9d5gk5/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: June 11, 2024
Last Modified: June 12, 2024
Protocol Integer ID: 101615
Keywords: malaria, Cas9, SHIFTiKO, Plasmodium
Funders Acknowledgements:
Marie Skłodowska Curie Individual Fellowship
Grant ID: 751865
Wellcome Trust
Grant ID: 220318/A/20/Z
European Society of Clinical Microbiology and Infectious Diseases
Grant ID: 2023
Abstract
This protocol describes a one-pot GoldenGate assembly of a new dual-guide targeting plasmid (pCas9-Duo) expressing two distinct guide RNAs in order to enhance the chances of a successful Cas9-mediated modification at a target region in Plasmodium falciparum. Designed as part of SHIFTiKO (frameshift-based trackable inducible knockout) system1 (based on2).
Attachments
pCas9-Duo.gb
20KB
Protocol materials
Nuclease-free Water
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
T4 Polynucleotide Kinase - 500 unitsNew England BiolabsCatalog #M0201S
Nuclease-free Water
UltraPure ATP 10mMPromegaCatalog #NC2683865
BbsI-HF - 300 unitsNew England BiolabsCatalog #R3539S
Cutsmart Buffer
Nuclease-free Water
T4 DNA LigaseRocheCatalog #11635379001
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
LB plates with 100 µg/ml ampicillin
M13 Reverse
M13Forward_reverse
gRNA oligo design
gRNA oligo design
Add the overhangs "ATTG" and "AAAC" to forward and reverse oligos of gRNA1, and "TTGG" and "TAAA" to gRNA2 respectively.
Double guide plasmid containing two gRNA expression cassettes
with distinct insertion sites.
Anneal gRNA oligos
Anneal gRNA oligos
1h 10m
1h 10m
Set up annealing reactions.
1.0 µL gRNA.F 100 micromolar (µM)
1.0 µL gRNA.R 100 micromolar (µM)
1.0 µL T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
0.5 µL T4 Polynucleotide Kinase - 500 unitsNew England BiolabsCatalog #M0201S
6.5 µL Nuclease-free WaterContributed by users
10m
Step 1 37 °C 00:30:00
Step 2 94 °C 00:05:00
Step 3 90 °C to 25 °C RAMP 5 °C per minute
Step 4 4 °C Hold
1h
Single-step GoldenGate assembly
Single-step GoldenGate assembly
1h 15m
1h 15m
Make 1:200 dilution mixture of annealed gRNA1 and gRNA2.
1 µL gRNA1 annealed reaction
1 µL gRNA2 annealed reaction
198 µL Nuclease-free WaterContributed by users
Set up assembly reaction
2 µL pCas9 200 ng/µL
1 µL 1:200 diluted oligo
0.5 µL BbsI-HF - 300 unitsNew England BiolabsCatalog #R3539S
0.5 µL T4 DNA LigaseRocheCatalog #11635379001
2 µL Cutsmart BufferContributed by users
2 µL UltraPure ATP 10mMPromegaCatalog #NC2683865
12 µL Nuclease-free WaterContributed by users
Step 1 37 °C 00:05:00
Step 2 16 °C 00:05:00
Repeat Step 1-2 for 6 cycles
Step 3 4 °C Hold
1h
Transform, plate and screen colonies
Transform, plate and screen colonies
20m 40s
20m 40s
Add 2 µL of assembly reaction to 15 µL ultracompetent cells (like XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314 ).
Place On ice for 00:20:00 .
20m
Heat shock at 42 °C for 00:00:40 and spread transformed cells onLB plates with 100 µg/ml ampicillinContributed by users .
40s
Pick colonies, miniprep-isolate plasmids and screen for gRNA1 and gRNA2 insertions by Sanger sequencing using M13 Reverse (CAGGAAACAGCTATGAC) and M13Forward_reverse (ACTGGCCGTCGTTTTAC), respectively.
Protocol references
Protocol developed as part of
1. Ramaprasad, Abhinay, and Michael J. Blackman. 2024. ‘A Scaleable Inducible Knockout System for Studying Essential Gene Function in the Malaria Parasite’. bioRxiv. https://doi.org/10.1101/2024.01.14.575607.
Based on
2. Adikusuma, Fatwa, Chandran Pfitzner, and Paul Quinton Thomas. 2017. ‘Versatile Single-Step-Assembly CRISPR/Cas9 Vectors for Dual gRNA Expression’. PLoS ONE 12 (12): e0187236. https://doi.org/10.1371/journal.pone.0187236.