Study of CRISPR/Cas9-induced genomic alterations as a result of targeted therapeutic genome editing.

Responsable du Stage : Mario Amendola, Alexandra Tachtsidi

E-mail: and


Résumé du Projet de Stage 

CRISPR/Cas9 technology has revolutionized our ability of genome editing and is a very promising tool for gene therapy. We have previously described (Pavani et al, 2020; Pavani et al, 2021) a novel ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using the CRISPR/Cas9 system, we integrated different transgenes under the transcriptional control of the endogenous α-globin promoter and achieved their high expression. With this approach we aim at developing curative strategies for monogenetic diseases. Before moving to the clinic, we need to further investigate the different potential outcomes of CRISPR/Cas9-based genome editing. It has been shown that Cas9-based genome editing can result into extensive on-target genome damage, including large deletions and complex genomic rearrangements.

Therefore, we aim at evaluating the structural variations of the targeted locus resulting from our genome editing approach. We want to explore the possible genomic alterations, i.e large deletions, insertions, translocations, that occur. To this purpose, we will be using third generation sequencing, a novel technology that allows to sequence long DNA fragments. We will perform long read sequencing (using the Oxford Nanopore Technology) of our region of interest that will be enriched from heigh molecular weight (HMW) genomic DNA using the CRISPR/Cas9-based enrichment procedure. From the sequencing data, we expect to have a profiling (i) of the different outcomes of the genome editing on our target site and (ii) of the therapeutic knock-in integration outcomes.

During the internship the master student will have hands-on cell culture and molecular biology techniques. The student will participate in the preparation and optimization of sequencing libraries, long read sequencing with the MinION/GridION Nanopore sequencer (in house), data collection and analysis.


Main tasks

Cell culture

Molecular biology techniques (DNA extraction, PCR, in vitro digestion, etc)

CRISPR techniques (gRNA design and testing, application)

Sequencing library preparation (protocol optimization and application)

Long read sequencing with Oxford Nanopore Technology MinION and/or GridION

Sequencing data analysis

References et publications récentes de l’équipe

Pavani G, Laurent M, Fabiano A, Cantelli E, Sakkal A, Corre G, Lenting PJ, Concordet J-P, Toueille T, Miccio A, Amendola M. (2020). Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins. Nat Commun. Jul 29;11(1):3778.

Pavani G, Fabiano A, Laurent M, Amor A, Cantelli E, Chalumeau A, Maule G, Tachtsidi A, Concordet J-P, Cereseto A, Mavilio F, Ferrari G, Miccio A, Amendola M. (2021). Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells. Blood Adv. Mar 9;5(5):1137-1153.

Gilpatrick T, Lee I, Graham JE, Raimondeau E, Bowen R, Heron A, Downs B, Sukumar S, Sedlazeck FJ, Timp W. (2020). Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol Apr;38(4):433-438.

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Intitulé de l’Unité : INTEGRARE UMR951

Nom du Responsable de l’Unité : Anne Galy

Nom du Responsable de l’Équipe : Mario Amendola

Adresse :  1 bis rue de l’Internationale, Evry