Dec 24, 2025
Version 2
Single-step assembly of double guide plasmid (pCas9-Duo) for gene-editing in Plasmodium V.2
- 1Francis Crick Institute;
- 2School of Infection and Immunity, University of Glasgow
Protocol Citation: Abhinay Ramaprasad 2025. 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/v2Version created by Abhinay Ramaprasad
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 24, 2025
Last Modified: December 24, 2025
Protocol Integer ID: 235780
Keywords: malaria, Cas9, SHIFTiKO, Plasmodium, editing in plasmodium, targeting plasmid, plasmodium falciparum, plasmodium, step assembly of double guide, distinct guide rna, distinct guide rnas in order, mediated modification, successful cas9, pcas9, double guide, editing
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
MRC Career Development Award
Grant ID: MR/Z504762/1
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
T4 DNA Ligase Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0202S
T4 Polynucleotide Kinase - 500 unitsNew England BiolabsCatalog #M0201S
Nuclease-free Water
LB plates with 100 µg/ml ampicillin
UltraPure ATP 10mMPromegaCatalog #NC2683865
BbsI-HF - 300 unitsNew England BiolabsCatalog #R3539S
Cutsmart Buffer
Hi-T4 DNA LigaseNew England BiolabsCatalog #M2622S
NEB 5-alpha Competent E.coli (High Efficiency) - 6x0.2 mlNew England BiolabsCatalog #C2987I
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314
M13 Reverse
M13Forward_reverse
gRNA oligo design
Add the overhangs "ATTG" and "AAAC" to forward and reverse oligos of gRNA1, and "TTGG" and "TAAA" to gRNA2 respectively. For predicting gRNAs, please refer to this protocol -
Protocol
CREATED BY
Abhinay Ramaprasad
Double guide plasmid containing two gRNA expression cassettes
with distinct insertion sites.
Note
When you have lot of targets, you can also use this sheet to create final sequences ready for oligo ordering to avoid inadvertent errors - 
pCas9-Duo-overhangs.xlsx12.6KB
pCas9-Duo-overhangs.xlsx12.6KB Anneal gRNA oligos
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 Water
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
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 Water
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 Hi-T4 DNA LigaseNew England BiolabsCatalog #M2622S
2 µL Cutsmart Buffer
2 µL UltraPure ATP 10mMPromegaCatalog #NC2683865
12 µL Nuclease-free Water
Step 1 37 °C 00:05:00
Step 2 25 °C 00:05:00
Repeat Step 1-2 for 6 cycles
Step 3 4 °C Hold
1h
Transform, plate and screen colonies
20m 40s
Add 2 µL of assembly reaction to 15 µL ultracompetent cells (like NEB 5-alpha Competent E.coli (High Efficiency) - 6x0.2 mlNew England BiolabsCatalog #C2987I ).
Note
We have observed that the competent cells used in the previous version -
XL-10 Gold Ultracompetent cellsAgilent TechnologiesCatalog #200314 do not perform consistently for this reaction.
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 ampicillin .
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.
Expected result
Usually we get 2-18 colonies out of which 2 colonies are screened.
Note
If processing a lot of samples, you can use this Shiny app pCas9-Duo-check to quickly screen the presence of both gRNA inserts in your colonies. Just ensure that you enter sample and primer names when submitting for Sanger sequencing such that your .ab1 filenames will be a) named uniquely for each colony and b) contain "M13R" or "M13FR" as primer names.
For some targets, we have observed that whilst gRNA01 insertion is very efficient, gRNA02 repeatedly fails. In these cases, instead of repeating the reaction from scratch or screening more colonies, we have had more success by simply repeating the GoldenGate reaction (Step 4 onwards) with pCas9-Duo-gRNA01 plasmid obtained in Step 10 as backbone and 1:200-diluted annealed gRNA02 as insert.
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.