The TOP vector: a new high-titer lentiviral construct for delivery of sgRNAs and transgenes to primary T cells.

ABSTRACT: Efficient delivery of nucleic acids for the engineering of primary T cells is central to the study of the basic biology of these key immune effector cells and has clinical implications. To date, lentiviral vectors delivering guide RNAs for CRISPR-Cas9 editing are not optimal for use in primary cells. Herein, we describe the T cell optimized for packaging (TOP) vector for delivering guide RNAs and transgenes into primary T cells. The TOP vector produces high-titer virus compared to a routinely used guide RNA vector, resulting in a ~10-fold increase in transduction in T cells. Moreover, a TOP vector expressing a chimeric antigen receptor and a guide RNA targeting the T cell receptor showed an ~5- to 9-fold increased transduction efficiency with ~2- to 3-fold higher expression compared to the commonly used epHIV7 vector and was simultaneously able to mediate efficient knockout of the endogenous T cell receptor in >71% of transduced cells upon Cas9 electroporation. The increased packaging of the TOP vector genome into viral particles appears to contribute to its higher transduction efficiency. The TOP vector represents an optimal tool for tandem delivery of transgenes and guide RNAs to primary T cells for use in functional screens and immunotherapy applications.

SUBMITTER: Humes D 

PROVIDER: S-EPMC7732963 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature