CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Summary

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

Abstract

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

Introduction

Malaria is a devastating disease caused by protozoan parasites of the genus Plasmodium. P. falciparum, the most deadly human malaria parasite, causes approximately 445,000 deaths per year, mostly in children under the age of five¹. Plasmodium parasites have an intricate life cycle involving a mosquito vector and a vertebrate host. Humans first become infected when an infected mosquito takes a blood meal. Then, the parasite invades the liver where it grows, develops, and divides for approximately one week.After this process, the parasites are released into the bloodstream, where they undergo asexual replication in red ....

Protocol

Continuous culture of P. falciparum requires the use of human RBCs, and we utilized commercially purchased units of blood that were stripped of all identifiers and anonymized. The Institutional Review Board and the Office of Biosafety at the University of Georgia reviewed our protocols and approved all protocols used in our lab.

1. Choosing a gRNA Sequence

1. Go to CHOPCHOP (http://chopchop.cbu.uib.no/) and select 'Fasta Target'. Under 'Target', paste the 200 base pairs from the 3’ end of the open reading frame (ORF) of a gene and 200 base pairs from the start of the gene’s 3&#....

Results

A schematic of the plasmids used in this method as well as an example of a shield mutation are shown in Figure 1. As an example of how to identify mutant parasites after transfection, results from PCRs for checking integration of the HA-glmS construct are shown in Figure 2. A representative image of a cloning plate is shown in Figure 3 to demonstrate the color change of the medium in the pre.......

Discussion

The implementation of CRISPR/Cas9 in P. falciparum has both increased the efficiency of and decreased the amount of time needed for modifying the parasite's genome, compared to previous methods of genetic manipulation. This comprehensive protocol outlines the steps necessary for generating conditional mutants using CRISPR/Cas9 in P. falciparum. While the method here is geared specifically for the generation of HA-glmS mutants, this strategy can be adapted for a variety of needs, including t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gene Pulser Xcell Electroporator	Bio-Rad	1652660
Gene Pulser Xcell Electroporator	Bio-Rad	165-2086	We buy the ones that are individually wrapped
Sodium Acetate	Sigma-Aldrich	S2889-250g
DSM1	Gift from Akhil Vaidya lab	Ganesan et al. Mol. Biochem. Parasitol. 2011 177:29-34
TPP Tissue Culture 6 Well Plates	MIDSCI	TP92006
TPP 100 mm Tissue Culture Dishes (12 mL Plate)	MIDSCI	TP93100
TPP Tissue Culture 96 Well Plates	MIDSCI	TP92096
TPP Tissue Culture 24 Well Plates	MIDSCI	TP92024
NEBuffer 2	New England Biolabs	#B7002S
NEBuffer 2.1	New England Biolabs	#B7202S
BtgZI	New England Biolabs	#R0703L
SacII	New England Biolabs	#R0157L
HindIII-HF	New England Biolabs	#R3104S
Afe1	New England Biolabs	#R0652S
Nhe1-HF	New England Biolabs	#R3131L
T4 DNA Polymerase	New England Biolabs	#M0203S
500 mL Steritop bottle top filter unit	Millipore	SCGPU10RE	You can use any size that fits your needs
EGTA	Sigma	E4378-100G
KCl	Sigma-Aldrich	P9333-500g
CaCl2	Sigma-Aldrich	C7902-500g
MgCl2	Sigma-Aldrich	M8266-100g
K2HPO4	Fisher	P288-500
HEPES	Sigma-Aldrich	H4034-500g
pMK-U6	Generated by the Muralidharan Lab	n/a
pHA-glmS	Generated by the Muralidharan Lab	n/a
pUF1-Cas9	Gift from the Jose-Juan Lopez-Rubio Lab	Ghorbal et al. Nature Biotech 2014
Glucose	Sigma-Aldrich	G7021-1KG
Sodium bicarbonate	Sigma-Aldrich	S5761-500G
Sodium pyruvate	Sigma-Aldrich	P5280-100G
Hypoxanthine	Sigma-Aldrich	H9636-25g
Gentamicin Reagent	Gibco	15710-064
Thymidine	Sigma-Aldrich	T1895-1G
PL6-eGFP BSD	Generated by the Muralidharan Lab
Puf1-cas9 eGFP gRNA	Generated by the Muralidharan Lab
NucleoSpin Gel and PCR Clean-up	Macherey-Nagel	740609.250
Albumax I	Life Technologies	N/A	You will want to try a few batches to find out what the parasites will grow in best
Human Red Blood Cells	Interstate Blood Bank, Inc	Email or call them directly for ordering	We typically use O+ blood
3D7 parasite line	Available upon request	N/A
Lysogeny Broth (LB)	Fisher	BP1426-2	You can make your own, it is not necessary to use exactly this
Ampicilin	Fisher	BP1760-25	We make a 1000X stock at 100mg/ml in water and store in the -20C
Ampicilin	Clonetech	R050A
Anti-EF1alpha	Dr. Daniel Goldberg's Lab	Washington University in St. Louis	You can use your preferred loading control for western blots. This is just the one we use in our laboratory
Rat Anti-HA Clone 3F10, monoclonal	Made by Roche, sold by Sigma	11867423001	You can use your preferred anti-HA antibody
0.6 mL tubes	Fisher	AB0350
Fisher HealthCare* PROTOCOL* Hema 3* Manual Staining System (Fixative+Solution I and II)	Fisher	22-122-911	You can also use giemsa stain
Fisherfines Premium Frosted Microscope Slides - Size: 3 x 1 in.	Fisher	12-544-3