Ligase I and Ligase III Mediate the DNA Double-Strand Break Ligation in Alternative End-Joining

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

Guangqing Lua,b,1, Jinzhi Duana,b,1, Sheng Shua,b, Xuxiang Wangb, Linlin Gaob, Jing Guoa,b, and Yu Zhanga,b,2

aGraduate School of Peking Union Medical College, Beijing 100730, China; and bNational Institute of Biological Sciences, Beijing 102206, China

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved December 22, 2015 (received for review November 2, 2015) In eukaryotes, DNA double-strand breaks (DSBs), one of the most DSBs. Both V(D)J recombination and class switching recom- harmful types of DNA damage, are repaired by homologous repair bination (CSR) have been used as important in vivo models to (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells study the NHEJ (7). During the V(D)J recombination, DSBs deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), generated by recombination-activating gene 1 (RAG1)/RAG2 substantial end-joining activities have been observed in various situ- endonuclease are joined exclusively by NHEJ. Deficiency of core ations, suggesting the existence of alternative end-joining (A-EJ) ac- NHEJ factors in mice, such as Lig4 and XRCC4, leads to com- tivities. Several putative A-EJ factors have been proposed, although plete abolishment of V(D)J recombination and lack of mature B results are mostly controversial. By using a clustered regularly inter- and T cells (8, 9). When combined with a checkpoint-deficient − − spaced short palindromic repeats (CRISPR)/CRISPR-associated protein genetic background (e.g., p53 / ), unresolved RAG-generated 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in DSB ends in B and T cells in core NHEJ factor-deficient mice addition to Lig4, either Lig1 or nuclear Lig3, representing the cells could result in dramatic genomic instability. In particular, on- containing a single DNA ligase (Lig3 or Lig1, respectively) in their cogenic chromosome translocations lead to the development of nucleus, was completely ablated. Surprisingly, we found that both lymphomaandleukemiainthosemice(10,11).Thefactthatthose Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ chromosome translocations are formed by end-joining in the ab- for class switching recombination (CSR) in the IgH locus and chromo- sence of core NHEJ factors suggested the existence of alternative somal deletions between DSBs generated by CRISPR/Cas9 in cis-chro- end-joining pathway(s) (A-EJ). CSR, by which mammalian mature mosomes. However, only deletion of nuclear Lig3, but not Lig1, could B cells change their production of Ig constant region type from one − − significantly reduce the interchromosomal translocations in Lig4 / to the other, represents an excellent model to study classic NHEJ cells, suggesting the unique role of Lig3 in catalyzing chromosome (c-NHEJ) and A-EJ (12, 13). DSBs in the IgH class switching (S) translocation. Additional sequence analysis of chromosome transloca- regions induced by activation-induced cytidine deaminase (AID) tion junction microhomology revealed the specificity of different are repaired by NHEJ via a loop-out and deletion mechanism (14). ligase-containing complexes. The data suggested the existence of In the absence of c-NHEJ core factors (such as Lig4, XRCC4, and multiple DNA ligase-containing complexes in A-EJ. Ku70/80), significant CSR activities, mediated by A-EJ, have been observed in both animals and cell lines (12, 13). There is no doubt DNA ligase | alternative end-joining | class switching recombination | that A-EJ contributes to all end-joining activities in the absence of chromosome translocation c-NHEJ. However, the contribution of A-EJ in the presence of c-NHEJ is still debatable. For example, it has been suggested that ammalian genomes are subjected substantial DNA damage A-EJ is the main end-joining activity to catalyze chromosomal Mfrom both endogenous processes [e.g., DNA replication, translocations in murine (15) but not in human cells (16). class switching recombination (CSR), etc.] and exogenous resources Although A-EJ activities have been observed in many cell (e.g., ionic radiation, DNA-damaging chemicals, etc.). Evolu- types and biological processes (12, 17–19), A-EJ’s exact com- tionarily conserved DNA repair pathways are essential to main- ponents and mechanisms have been still not clearly revealed and tain both the structure integrity and the information accuracy of the genome (1). In eukaryotes, DNA double-strand breaks (DSBs), Significance one of the most dangerous and severe types of DNA damage, are repaired mainly by two evolutionarily conserved repair pathways: DNA repair is not only extremely important for the genome homologous repair (HR) and nonhomologous end-joining (NHEJ) stability in normal cells but also extensively involved in carci- (2). NHEJ directly ligates two broken DSB ends, whereas HR uses nogenesis and evolution. DNA double-strand breaks (DSBs), a homologous template (in most of cases, the sister chromosome) one of the most harmful types of DNA damage, are repaired for repair. Although NHEJ is simpler and faster than HR, repair mainly by homologous repair (HR) and nonhomologous end- by NHEJ often leads to change of sequences in the repair junctions joining (NHEJ). Although the components of NHEJ have been via deletion, insertion, and mutations. Most importantly, NHEJ studied extensively in last two decades, in cells deficient for can also directly ligate two distant DSBs (both in cis-andtrans- core classic NHEJ factors, significant DSB end-joining activities chromosomes), therefore leading to chromosomal deletions and have been observed in various situations, suggesting the ex- translocations that are tightly linked to the genome evolution and istence of the alternative end-joining (A-EJ) activities. Here, we carcinogenesis (3). dissected the roles of DNA ligases in mediating the last step The NHEJ in eukaryotes has been extensively studied in the last of A-EJ. The results suggested the existence of multiple DNA two decades (4–6). Mechanistically, NHEJ could be separated into ligase-containing complexes in A-EJ. several steps. First, in the DSB binding and tethering step, the DSB ends are recognized and bound by Ku70/Ku80 heterodimers Author contributions: Y.Z. designed research; G.L., J.D., S.S., X.W., and J.G. performed that function as docking sites for other NHEJ factors. In the next research; G.L., J.D., and L.G. analyzed data; and Y.Z. wrote the paper. end-processing step, nuclease and DNA polymerase activities The authors declare no conflict of interest. are recruited to remove damaged or mismatched nucleotides and This article is a PNAS Direct Submission. prepare the broken ends for ligation. Finally, DNA ligase IV 1G.L. and J.D. contributed equally to this work. (Lig4) complex, consisting of DNA Lig4 and its cofactor X-ray 2To whom correspondence should be addressed. Email: [email protected]. repair cross-complementing protein 4 (XRCC4), as well as the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. newly identified XRCC4-interacting factor (XLF), reseals the 1073/pnas.1521597113/-/DCSupplemental.

1256–1260 | PNAS | February 2, 2016 | vol. 113 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1521597113 Downloaded by guest on September 24, 2021 sometimes are controversial (5, 20, 21). For example, whether A-EJ is a completely independent new pathway or an alternative c-NHEJ pathway in which alternative components could sub- stitute the missing c-NHEJ factors is still debatable. Comparing with large numbers of factors and pathways involved in the early DSB repair steps, there are only three known DNA ligases (DNA Lig1, DNA Lig3, and DNA Lig4) in mammalian cells to finish the last ligation step (22). It has been proposed that those three DNA ligases function differently in various DNA metabolism processes. Although all three mammalian DNA ligases have highly homologous catalytic cores (including DBD, AdD, and OB-Fold do- mains), through their distinct N- and C-terminal regions, the DNA ligases may interact with different partners, which could confer functional specificity. In DSB repair, the role of Lig4 has been mostly restricted to c-NHEJ, whereas both Lig1 and Lig3 have been suggested to mediate the A-EJ in vitro and in vivo (23–28). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to gen- erate cell lines in which Lig1 or Lig3 were completely depleted, and we tried to unequivocally reveal the ligases’ roles in A-EJ. Results Establishment of Mammalian Cell Lines Containing only a Single DNA Ligase in Nucleus. To unequivocally study the repair of endogenous chromosomal DSBs by the A-EJ pathway(s) in vivo, we used CH12F3 (CH12) cells, which are proficient or deficient of the core c-NHEJ factor Lig4 (13, 29). To completely inactivate Lig1 in those cells, CRISPR/Cas9 and two single-guide RNAs CELL BIOLOGY (sgRNAs) were designed to delete exons 18 and 19 of the mouse Lig1 gene (Fig. 1A). PCR strategy was used to identify the cell lines in which both alleles of Lig1 were deleted (Fig. 1C). Western blot results showed that there was no detectable Lig1 − − protein in Lig1 / cells (Fig. 1E). For Lig3 inactivation, exons – 8 13 of the Lig3 gene, which encode catalytic core of Lig3 pro- Fig. 1. Generation of mouse CH12 cells deficient for Lig1 and nuclear Lig3. tein, were deleted by Cas9 and two sgRNAs (Fig. 1B). Because (A and B) Schematic representation for the genomic organization of WT and the mitochondria isoform of Lig3 protein is essential for the CRISPR/Cas9/sgRNA-deleted Lig1 (A)andLig3 (B) loci. Target sites of CRISPR/ maintenance of normal mitochondrial function and cell viability, Cas9/sgRNA as well as primers used for genotyping are indicated. (C)PCR + + + − − − to deplete the nuclear form of Lig3, we had to first stably express genotyping of Lig1 / (lane 1), Lig1 / (lane 2), and Lig1 / (lane 3) cells. (D)PCR + + + − − − Δ genotyping of Lig3 / (lane 1), Lig3 / (lane 2), and Lig3 / (lane 3) cells. a MtLig3- BRCT-GFP-NES protein that locates exclusively in −/− mitochondria and cytoplasm but not in nucleus (30) (Fig. S1 and (E) Western blot analysis of Lig1 protein. In Lig1 cells (lane 1), the Lig1 protein is completely lost. Lane 2 is the WT control cell line. (F) Western blot Fig. 1D). Western blot and PCR genotyping were used to identify −/− analysis of Lig3 protein. In nuclear Lig3 cells (lane 1), the WT Lig3 protein the cell lines specifically deficient for nuclear Lig3 (Fig. 1 D and (∼105 kDa) is completely lost. In lane 1, the rescued Mtlig3-ΔBRCT-GFP-NES F). In the rest of this report, we simplified such nuclear Lig3- ∼ − − protein ( 130 kDa) is marked by an asterisk. Lane 2 is the WT control cell line. deficient cells as Lig3 / cells. From the Lig4-deficient cell line (13), we generated CH12 − − − − cells deficient for either both Lig1 and Lig4 (Lig1 / Lig4 / )or Deletion of Either Lig1 or Nuclear Lig3 Could Not Affect A-EJ– − − − − both Lig3 and Lig4 (Lig3 / Lig4 / )(Fig. S2). In other words, in Mediated IgH CSR. We examined the cytokine-induced IgH CSR − − − − Lig1 / Lig4 / cells, the only remaining nuclear DNA ligase is in various CH12 cell lines (Fig. 2 and Fig. S4). Surprisingly, − − − − Lig3, whereas in Lig3 / Lig4 / cells, the only nuclear DNA li- deletion of either Lig1 or nuclear Lig3 in Lig4-deficient cells + gase is Lig1. We also tried to obtain cell lines in which both Lig1 could not further reduce the percentage of IgA-positive (IgA ) + and nuclear Lig3 are deleted (i.e., a Lig4-only cell line). How- cells after CSR; these IgA cells represent the successful IgH ever, such cell lines could not be obtained (Table S1). − − CSR products catalyzed by A-EJ (Fig. 2A, Upper). At the same Comparing with Lig4 / cells, additional deletion of either − − −/− −/− −/− −/− time, the percentages of IgA and IgM double-negative (IgA IgM ) Lig1 (Lig1 Lig4 ) or nuclear Lig3 (Lig3 Lig4 ) did not cells, representing cells in which CSR failed after DSBs were in- − − − − − − significantly affect the cell proliferation without or with cytokine troduced in switching regions, are similar in Lig4 / ,Lig1 / Lig4 / , stimulation (Fig. S3 A and B, respectively) and did not affect the − − − − and Lig3 / Lig4 / cells (Fig. 2A, Lower). cells’ sensitivity to hydroxyurea (HU) (Fig. S3C), suggesting that To further refine the direct roles of Lig1 and Lig3 in the DSB Lig1 and Lig3 can completely replace each other’s function in repair step during CSR, we developed an AID-independent CSR DNA replication. However, both double-deficient cells showed μ α slightly but significantly more sensitivity to methyl methanesulfo- assay, in which Cas9/sgRNAs targeting S and S regions were nate (MMS) and PARP inhibitor (Olaparib) (Fig. S3 D and E, cotransfected into normal cultured CH12 cells without cytokine − − − − respectively). In addition, comparing with Lig4 / and Lig1 / stimulation (Fig. 2B and Fig. S5). Such Cas9/sgRNA-induced − − − − − − Lig4 / ,Lig3 / Lig4 / cells also showed slightly more sensitivity DSBs in class switching regions could be efficiently joined by to Zeocin (Fig. S3F). These similarities and differences in re- c-NHEJ and A-EJ activities in both wild-type (WT) and Lig4- sponse to various DNA-damaging reagents suggest that Lig1 and deficient CH12 cells (Fig. S5). However, the end-joining effi- Lig3 play both complementary and noncomplementary functions ciencies between such AID-independent DSBs within the IgH − − − − − − − − − − in DNA repair. locus in Lig4 / , Lig1 / Lig4 / , and Lig3 / Lig4 / cells are not

Lu et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1257 Downloaded by guest on September 24, 2021 − − − − − − − − − − A-EJ activities in Lig4 / ,Lig1 / Lig4 / , and Lig3 / Lig4 / cells are not significantly different (Fig. 3B). The above results further confirmed that Lig1 and Lig3 could completely replace each other’sroleindeletionalA-EJ.

Interchromosomal Translocations by A-EJ in the Absence of Either Lig1 or Nuclear Lig3. We set up a nested-PCR assay for chromosomal translocations between Cas9/sgRNA-induced DSBs in nonhomologous chromosomes (Fig. S6 A and B). Translocation frequencies between the Tet2 locus in chromosome 3 and the HDAC6 locus in chromosome X (Tet2/HDAC6;Fig.4A and Fig. S6C) and between the Tet2 locus and the ADA2 locus in chromosome 2 (Tet2/ADA2;Fig.4B and Fig. S6D) increased significantly in Lig4-deficient CH12 cells in comparison with WT. Further de- − − − − letion of Lig1 (Lig1 / Lig4 / ) could not affect the frequencies of both chromosome translocations. Surprisingly, deletion of nuclear − − − − Lig3 (Lig3 / Lig4 / ) resulted in a significant reduction of translocations catalyzed by A-EJ in Lig4-deficient cells. In other words, in − − − − Lig3 / Lig4 / cells, Lig1 could not fully replace the translocation − − − − ligation activities of nuclear Lig3. However, in Lig1 / Lig4 / cells, Lig3 could largely compensate the loss of Lig1 in chromosome translocations. These results suggested that for A-EJ activities mediating chromosome translocations, Lig1 and Lig3 have both complementary and noncomplementary functions. The individual Tet2/HDAC6 translocation junctions were isolated for sequence analysis. As shown in Fig. 4C, Table S2, and Dataset S1, the direct joints, which represent the direct ligation of DSB ends generated by precise cleavage of Cas9/sgRNA com- − − plexes, decreased significantly (10/62 in WT vs. 1/64 in Lig4 / ) when Lig4 was deleted. Therefore, DSBs without end-processing might the preferential substrates for Lig4 and c-NHEJ during chromosome translocation. On the other hand, it is also possible that c-NHEJ is kinetically faster than A-EJ and therefore will be used first before the significant DSB end-processing happens. We also compared the distribution of microhomologies in junctions isolated from various cell lines (Fig. 4D and Fig. S6F). As − − expected, the junctions in Lig4 / cells showed significantly longer microhomologies than in WT cells. In particular, the percentage of blunt junctions [without microhomology (MH = 0)] was re- − − duced from 50% in WT to 10% in Lig4 / cells. Interestingly, − − − − − − − − in Lig3 / Lig4 / but not in Lig1 / Lig4 / cells, such blunt Fig. 2. Deletion of either Lig1 or nuclear Lig3 could not affect A-EJ–medi- junctions are further reduced. At the same time, in Lig1-only ated CSR in Lig4-deficient CH12 cells. (A) Summary of FACS analysis results −/− −/− from cytokine-induced CSR in various CH12 cells after 48 h (Left) and 72 h (Lig3 Lig4 ) cells, translocation junctions have significantly + (Right). Results of surface staining of IgA positive (IgA ) cells (Upper) and longer microhomology. The results suggest that during A-EJ the − − IgA/IgM double-negative (IgA IgM ) cells (Lower) are shown. (B) Summary blunt junctions are likely preferential substrates of Lig3, whereas of FACS analysis results from Cas9/sgRNA induced CSR in various CH12 cells Lig1 ligates preferentially junctions with longer microhomologies. after 48 h (Lower Left) and 72 h (Lower Right). (B, Upper) Schematic representation of the assay and the localization of sgRNAs used. Mean per- Discussion centages of IgA+ or IgA−IgM− for each cell line from three independent −/− −/− DNA ligases catalyze the formation of a phosphodiester bond to experiments were calculated. For Lig1,4 and Lig3,4 cell lines, the results join broken DNA strands together and therefore play essential from three independent single clones were combined. Background from normal culture without cytokine was subtracted in all experiments. Error roles in nuclear DNA metabolism, including DNA replication, bars indicate the SD. base excision repair (BER), nucleotide excision repair (NER), single-strand break repair, mismatch repair, homology-mediated DSB repair, and NHEJ of DSB (22). Whereas the function of significantly different, suggesting such A-EJ activities are not Lig4 is mostly restricted to c-NHEJ, how Lig1 and Lig3 con- solely dependent on either Lig1 or Lig3. tribute to the other DNA repair processes has not been studied in details in vivo. We have generated various cell lines genetically Intrachromosomal DSB Deletional A-EJ in Lig1- or Nuclear Lig3- deficient for one or two DNA ligases; these cell lines provide Deficient CH12 Cells. CSR in the IgH locus may represent a spe- useful tools to clarify the in vivo functions and specificity of DNA cial form of A-EJ that is complicated by IgH locus-specific synapse ligases in nuclear DNA metabolism. formation, AID-induced DSBs, and end-processing (14, 31). In particular, both Lig1 and Lig3 have been proposed to me- Therefore, we further investigated the A-EJ activities between two diate A-EJ (23–28). By analyzing the A-EJ activities in cells − − − − − − − − non-IgH chromosomal DSBs. We designed four sgRNAs targeting containing only Lig1 (Lig3 / Lig4 / ) or Lig3 (Lig1 / Lig4 / ), different genomic loci in chromosome 8 (Fig. 3A). By cotrans- we found that both Lig1- and Lig3-containing complexes could fecting distinct Cas9/sgRNA pairs, the deletional end-joining be- efficiently catalyze A-EJ for chromosomal deletions. However, tween two sgRNA-induced DSBs separated by various distances for A-EJ–mediated interchromosomal translocations, Lig1- and (4, 40, and 370 kb) could be analyzed by quantitative real-time Lig3-containing complexes showed different activities. More PCR. The results showed that the efficiencies for those deletional specifically, deletion of nuclear Lig3 could significantly reduce

1258 | www.pnas.org/cgi/doi/10.1073/pnas.1521597113 Lu et al. Downloaded by guest on September 24, 2021 Fig. 3. Intrachromosomal DSB repair by A-EJ in Lig1- or nuclear Lig3-deficient CH12 cells. (A) Schematic representation of the intrachromosomal deletion assay. The CRISPR/Cas9 and sgRNAs were designed to introduce individual DSB spanning a 370-kb region on chromosome 8. (B) Summary of the quantitative real-time PCR analysis of intrachromosomal deletion efficiencies between two sgRNA-induced DSBs separating by various distances (4, 40, and 370 kb). Relative deletion frequencies in different cell lines were normalized to WT cells and also adjusted by transfection efficiency. Error bars indicate the SD of four independent experiments. For Lig1,4−/− and Lig3,4−/− cell lines, the results from three independent single clones were combined. CELL BIOLOGY

− − the translocation frequency in Lig4 / cells, whereas deletion of ends without end-processing are the preferential substrates for Lig1 could not. In addition, analysis of translocation junction Lig4 complex(s); during A-EJ, the blunt junctions without micro- microhomology revealed the specificity of different ligase com- homology are preferential substrates of Lig3 complex(s), whereas plexes. During chromosome translocation, chromosomal DSB Lig1-complex(s) ligates preferentially junctions with longer microhomologies. We propose that those three kinds of ligase complexes may compete for the final ligation step during chromosome translocation. The components of such ligase complexes are still waiting to be systematically characterized. Materials and Methods Plasmids. The pcDNA3.3-hCas9 and pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmids were obtained from Addgene (no. 41815 and no. 42230). The pCR- Blunt II-TOPO-sgRNA empty cloning vector was obtained from Addgene (no. 41824), and the sgRNAs for Lig1 and Lig3 deletions and for chromosome translocation assays were constructed following the Church Laboratory sgRNA Synthesis Protocol (www.addgene.org/static/data/93/40/adf4a4fe-5e77- 11e2-9c30-003048dd6500.pdf). Other sgRNA plasmids were constructed following the Zhang Laboratory gRNA Synthesis Protocol (www.genome-engineering.org/ crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509. pdf). Lig3-GFP and MtLig3-ΔBRCT-GFP-NES cDNAs were assembled as described by Simsek et al. (26) and cloned into the pPB-LR51-EF1α-puro2ACas9 (a gift from KosukeYusa, Wellcome Trust Sanger Institute, London). All PCR- generated fragments were verified by Sanger sequencing. See oligo information in Table S3.

Antibodies. Antibodies to Lig1 (18051-1-AP; Proteintech Group), Lig3 (611876; BD Biosciences), and α-tubulin (T6199; Sigma-Aldrich) were used in West- ern blot. FITC-conjugated anti-mouse IgA (559354; BD Biosciences) and allophycocyanin (APC)-conjugated anti-mouse IgM (17-5790-82; Affymetrix/ eBioscience) were used in FACS analysis.

Fig. 4. Interchromosomal translocation mediated by A-EJ in Lig1- or nuclear Cell Culture and Transfection. CH12 cells were cultured in RPMI 1640 medium × Lig3-deficient CH12 cells. (A and B) Summary of the relative chromosomal supplemented with 10% (vol/vol) FBS, 1 nonessential amino acids, 1 mM so- β translocation efficiencies of t(Tet2/HDAC6)(A) and t(Tet2/ADA2)(B). Error dium pyruvate, 20 mM Hepes, 2 mM L-glutamine, 50 mM -mercaptoethanol, − − bars indicate the SD (of four independent experiments). For Lig1,4 / and and 5% (vol/vol) NCTC-109; 1 μg of indicated plasmid was transfected into − − 6 Lig3,4 / cell lines, the results from three independent single clones were 1 × 10 CH12 cells following the NeonTransfection System protocol with combined. (C) Distribution of t(Tet2/HDAC6) translocation junctions. Per- 1,100 voltages, 20 microseconds, and 2 pulses. centages of direct joints (black), deletional junctions (red), and insertions − − − − (blue) were calculated for WT (n = 62), Lig4 / (n = 64), Lig1,4 / (n = 71), and Generation of Lig1 and Nuclear Lig3-Deficient Cell Lines. For Lig1 and Lig3 − − Lig3,4 / (n = 97) cell lines. (D) Distribution of t(Tet2/HDAC6) junction deletion, pcDNA3.3-hCas9, indicated sgRNA expression plasmids, and a ve- microhomologies in indicated cell lines. Junctions with insertions were ex- nus expression vector were cotransfected into CH12 cells; 72 h after trans- cluded. Mann–Whitney U test was performed. fection, venus-positive cells were sorted as single clones into 96-well plates

Lu et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1259 Downloaded by guest on September 24, 2021 by BD FACSAria II. Individual clones were genotyped by PCR, and positive Cas9/sgRNA-Induced Chromosomal Deletion Assay. Atotalof1× 106 CH12 clones were verified by Western blot. cells were electroporated with indicated sgRNA and Cas9 vectors. For Lig3 deletion, before Cas9/sgRNA deletion, cell lines stably expressing Chromosomal deletion frequency was determined by quantitative real- Δ Lig3-GFP and MtLig3- BRCT-GFP-NES proteins were established by the pig- time PCR using GAPDH as an internal control using genomic DNA iso- gyBac transposon system, as described previously (32). See oligo information lated after 72 h posttransfection. Relative deletion frequencies were in Table S3. also normalized to transfection efficiency. See oligo information in Table S3. Cell-Proliferation and Drug-Sensitivity Assays. To monitor cell proliferation and drug sensitivity, indicated cells were seeded at 5 × 104 cells/mL with various Cas9/sgRNA-Mediated Chromosomal Translocation. For translocations, 2 × 106 chemicals added at different concentrations in a 96-well plate and cultured for of CH12 cells were electroporated with indicated sgRNA and Cas9 vectors. 24 or 48 h. Then, 20 μL of reconstituted CellTiter-Glo reagent (Promega G7570) with 80 μL of cell culture medium was added to each well. The contents were Genomic DNAs were collected at 72 h posttransfection. Chromosomal mixed on an orbital shaker for 5 min to induce cell lysis and were subsequently translocations were detected and quantified by a nested-PCR approach. The incubated at room temperature for 5 min to stabilize the luminescent signal, results were also normalized to transfection efficiency. Individual chromo- which was recorded using a Multimode Plate Readers (2300-001M; PerkinElmer). somal translocation junctions were sequenced to verify translocations and determine the junction sequences. See oligo information in Table S3. Cytokine-Stimulated and Cas9/sgRNA-Induced CSR Assays. For cytokine-induced CSR assay, CH12 cells were seeded at 5 × 104 cells/mL in the presence of 1 ng/mL ACKNOWLEDGMENTS. We thank members of the Y.Z. laboratory for anti-CD40 (16-0402-86; eBioscience), 5 ng/mL of IL-4 (404-ML; R&D Systems), and helpful discussions and support. This research was supported by the 0.5 ng/mL TGF-β1 (240-B; R&D Systems) and cultured for 48 and 72 h. Program for Excellent Talents by the Beijing municipal government (Grant For Cas9/sgRNA-induced CSR assay, 1 × 106 CH12 cells were transfected 2013D008013000002); the Hundred, Thousand, and Ten Thousand Talent Project by the Beijing municipal government (Grant 2015001); the National with 0.5 μg of pX330-Sμ-sg and 0.5 μg of pX330-Sα-sg and cultured for 48 Thousand Young Talents Program of China; and the National Natural and 72 h. Cells were stained with FITC-conjugated anti-mouse IgA and APC- Science Foundation of China (Grant 81572795) (to Y.Z.). We thank the conjugated anti-mouse IgM antibodies and then analyzed with an Accuri C6 municipal government of Beijing and the Ministry of Science and Tech- Flow Cytometer (BD Biosciences). CSR efficiencies were determined as the nology of China for funds allocated to National Institute of Biological percentage of IgA-positive cells. Sciences (NIBS).

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