Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

Timothy J Satchwell, Katherine E Wright, Katy L Haydn-Smith, Fernando Sánchez-Román Terán, Joseph Hawksworth, Jan Frayne, Ashley M Toye, Jake Baum

Preprint posted on 13 December 2018

Article now published in Nature Communications at

CRISPR meets red blood cells – a new model system to illuminate the roles of host proteins in malaria parasite invasion

Selected by Alyson Smith

Why I think this study is interesting

This study turns over a new leaf in malaria research by applying a new model system to directly test the roles of red blood cell proteins in the parasite life cycle. Modern genetic technologies (e.g., CRISPR-Cas9) can now be employed to answer questions that were previously difficult or impossible to address. This and future studies can accelerate the development of therapies for this serious disease and answer other standing questions in basic and translational red blood cell biology.


Parasites of the Plasmodium genus cause malaria by invading red blood cells and developing within them, ultimately causing red blood cell membrane rupture (1). Mature human red blood cells and reticulocytes (their immediate precursors in erythroid differentiation) lack nuclei, preventing genetic manipulation to probe the roles of host proteins in Plasmodium infection. While malaria researchers have used proteases, antibodies, and rare spontaneous mutations to study this process, our knowledge of host protein involvement remains incomplete.

To address this issue, scientists have developed in vitro culture systems using primary hematopoietic stem cells (CD34+ cells) or immortalized erythoid cell lines. However, CD34+ cells proliferate only briefly in an undifferentiated state and immortalized cell lines do not efficiently produce anucleate reticulocytes. These factors limit the efficiency of production of genetically modified reticulocytes to use in malaria invasion models.

Some of the authors of this study recently developed an immortalized human adult erythroid cell line (Bristol Erythroid Line Adult, or BEL-A) that can proliferate in an undifferentiated state and can be induced to terminally differentiate and form anucleate reticulocytes (2). In this study, the authors use this cell line and CRISPR-Cas9 to test the role of host proteins in P. falciparum invasion.

Key findings

Bristol Erythroid Line Adult (BEL-A)-derived reticulocytes are an effective model for P. falciparum invasion. 

The authors induced terminal differentiation in BEL-A cells and isolated anucleate reticulocytes. After infection with P. falciparum, these reticulocytes had similar parasitemia to native, mature red blood cells. The parasite appeared to grow, mature, and reinvade at similar rates in both systems.

Basigin (BSG) is required for P. falciparum invasion, but this activity does not require the BSG cytoplasmic tail. 

Previous work has implicated BSG (a host surface receptor) in the invasion process, and in other cell types the cytosplasmic tail of BSG plays a role in signaling. Using CRISPR-Cas9, the authors knocked out BSG without affecting BEL-A expansion, terminal differentiation, or expression of known P. falciparum receptors (GPA, GPC, band 3, CD55, and CD44). Both wild type BSG and BSG with the cytoplasmic tail truncated (expressed at close to endogenous levels) rescued P. falciparum invasion on the BSG KO background. These experiments confirm the role of BSG in invasion, but future work is required to find the BSG domain responsible for this activity.

Cyclophilin B (CypB) is not required for P. falciparum invasion.

A recent study suggested that CypB forms a multiprotein complex (including BSG) that is required for invasion. Using CRISPR-Cas9, the authors found that CypB KO BEL-A-derived reticulocytes had similar parasitemia to unmodified reticulocytes, suggesting that CypB is not required for invasion.

Flow cytometry can detect parasitemia in BEL-A-derived reticulocytes.

In mature red blood cells – which lack DNA and mRNA – parasitemia can be quickly measured using nucleic acid-binding dyes to detect parasite DNA by flow cytometry. In this study, the mRNA in the BEL-A-derived reticulocytes and contaminating DNA from nucleated reticulocyte precursors and unruptured schizonts created significant background in this assay. Despite this, the authors detected differences in parasitemia between BSG KO and wild type reticulocytes. The authors recommend using light microscopy to confirm phenotypes detected in flow cytometry.

Future directions

This study opens the door to future work to elucidate the roles of host proteins in P. falciparum invasion, development and egress. Because BEL-A-derived reticulocytes express receptors for other Plasmodium species (P. vivax and P. knowlesi), this system can be expanded to study these species as well. Because flow cytometry can detect changes in parasitemia in this system, it can be adapted for high-throughput screens to test the roles of host proteins and develop new therapeutics.

Despite the utility of BEL-A-derived reticulocytes as a model system, they likely fail to reproduce all aspects of native Plasmodium infection. The malaria community will have to devise methods to improve this system and verify its results. Beyond malaria research, the genetic toolkit available in other human cell lines can now be applied to expand our understanding of reticulocyte and erythroid biology and pathology.


  1. Koch M & Baum J (2016) The mechanics of malaria parasite invasion of the human erythrocyte – towards a reassessment of the host cell contribution. Cell Microbiol 18(3):319-329.
  2. Trakarnsanga, K., Griffiths, R.E., Wilson, M.C., Blair, A., Satchwell, T.J., Meinders, M., Cogan, N., Kupzig, S., Kurita, R., Nakamura, Y., et al. (2017). An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nature communications 8, 14750.

Tags: crispr, malaria, parasite invasion, red blood cells

Posted on: 8 January 2019


Read preprint (2 votes)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage