In vitro and in vivo whole genome CRISPR screening under drug treatment in T-ALL

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Introduction: Treatment resistance is a pivotal problem in T-ALL, and identifying the targets involved is key to improving therapy. Currently dexamethasone, daunorubicin, vincristine and asparaginase form the basis of UK T-ALL induction therapy. Methods: We are establishing a protocol to perform a whole genome CRISPR screen in vitro and in vivo in the presence of chemotherapeutics using the GeCKOv2 library. Karyotype, cytogenetic background and transduction efficiencies of T-ALL cell lines are examined to ensure successful knockout and sufficient library coverage. In vitro sensitivity of T-ALL cell lines to chemotherapeutics are assessed by resazurin assay. For in vivo screening T-ALL cell lines are injected intrafemorally into RAG2-/-gc-/- (RG) mice. We assess transplantable cell numbers, time to and pattern of engraftment. Upon drug dosing, toxicity, pharmacokinetics, and changes in leukaemic burden will be assessed. Results: In vitro HPB-ALL (pseudodiploid with TLX3 translocation) is sensitive to asparaginase, daunorubicin and vincristine, but resistant to dexamethasone. HPB-ALL engrafts in RG mice and leukaemic progression can be monitored by in vivo bioluminescence imaging.

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