For non-reducing condition, supernatants were mixed with loading buffer without reducing reagents. normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. CRISPR-Cas9 has been used to generate a range of genetic modifications including gene knock-outs and chromosomal rearrangements. Here the authors target the ACY-1215 (Rocilinostat) immunogloblin genes and demonstrate the induction of class switch recombination, opening up possibilities for research and antibody production. Gene 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 step1,2,3. In physiological conditions, DSBs are launched at the Ig genes by the activity of B-cell-specific enzymes such as recombination-activating gene 1/2 (RAG1/2) and activation-induced cytidine deaminase (AID)1,2,3. During CSR, AID generates DSBs in the Ig locus by targeting repetitive sequences in the switch (S) regions that precede each Ig heavy (IgH) coding sequence1,2,3. Paired DSBs in the ACY-1215 (Rocilinostat) switch regions are then joined by the classical and alternative non-homologous end-joining (NHEJ) pathways to generate a switch of the IgH4. This long range joining is usually thought ACY-1215 (Rocilinostat) to be part of a general mechanism of DNA repair where two DSBs are joined incisover long chromosome distances5. Indeed, efficient CSR can be obtained in absence of AID or S regions after the introduction of DSBs by site-specific I-SceI endonuclease6. The bacterial type II clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 (CRISPR-associated Protein 9) systems have great potentials for RNA-guided genome editing, including multiplexing genome engineering, gene targeting by homologous recombination, regulation of transcription, chromosomal translocation formation, high-throughput functional genomic screens and even RNA editing7,8,9,10. We as well as others exhibited that when two DSBs are simultaneously launched in a cellin vitroorin vivoby CRISPR-Cas9 activity, a variety of gene rearrangements are generated, including large deletions (up to 12 Mb), inversions and chromosomal translocations11,12,13,14. Here we show that this CRISPR-Cas9 system can be adapted to efficiently induce CSR in main mouse B cells and human B-cell lines. In addition, this system can be proficiently used to ACY-1215 (Rocilinostat) engineer hybridoma cells to produce monoclonal antibody with a switched Ig heavy chain or to secrete the immunoglobulin Rabbit polyclonal to ZFYVE9 Fab fragments only. == Results == == CRISPR-Cas9-mediated CSR in ACY-1215 (Rocilinostat) mouse B cells == Since CSR is usually a DNA deletion induced by two DSBs occurring in the S regions preceding the IgH constant sequences, we sought to engineer CSR by CRISPR-Cas9-mediated DNA deletion. We first designed a system to target the mouseIgHlocus. Given that S regions are highly repetitive, we generated lentiviral vectors expressing Cas9 and guide-RNA (gRNA) targeting the more specific DNA sequences flanking immediately upstream (S 5 gRNA and S1 5 gRNA) or downstream (S 3 gRNA and S1 3 gRNA) of the S regions that precede the mouse C and C1 IgH constant sequences (Fig. 1aandSupplementary Fig. 1a). Efficiency of selected gRNA sequences was tested by Surveyor assay (Supplementary Fig. 1b). By introducing simultaneous DSBs in sites flanking the S and S1 regions, the prediction was to generate deletions of the DNA segment encompassed by the two DSBs as well as excision circles in a process closely mimicking CSR occurring in B cells3. To test this prediction, we first transduced immortalized mouse fibroblasts. PCRs with specific primers confirmed that this expected deletions were obtained with all four gRNA combinations (Fig. 1a). Sanger sequencing of PCR products exhibited not only the expected DNA junctions with a predominance of precise junctions between the Cas9-mediated DSBs, but also 5 or 3 deletions and insertions, as previously described11,12,13,14(Fig. 1b). PCR also detected the expected excision circles as well as inversions of the DNA segment encompassed by the DSBs, as we previously exhibited in a different model13(Supplementary Fig. 2a,b). == Physique 1. Induction of class-switch recombination (CSR) by CRISPR-Cas9 system in mouse cells. == (a) Top: genomic business of the mouseIgHconstant region locus and position of the.