Abstract
Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.
Highlights
DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings
Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired Ig heavy (IgH) switching helpful to study the biology of normal and lymphoma B cells
double-strand breaks (DSBs) are introduced at the Ig genes by the activity of B-cell-specific enzymes such as recombinationactivating gene 1/2 (RAG1/2) and activation-induced cytidine deaminase (AID)[1,2,3]
Summary
DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. We show the application of CRISPR-Cas[9] technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas[9] and guide-RNA (gRNA) with retro- or lenti-virus to IgM þ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. We induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Correspondence and requests for materials should be addressed to R.C. G ene rearrangements editing the immunoglobulin (Ig) genes such as V(D)J recombination and class-switch recombination (CSR) require the formation of DNA double-strand breaks (DSBs) as the key initiating step[1,2,3].
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