C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

Article26 April 2022Open Access Source DataTransparent process C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency Xia Xie Xia Xie orcid.org/0000-0002-0423-976X State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Tingting Gan Tingting Gan orcid.org/0000-0002-4679-9212 The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization, Formal analysis, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Bing Rao Bing Rao orcid.org/0000-0001-9895-4133 State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Formal analysis, ​Investigation, Writing - review & editing Search for more papers by this author Weiwei Zhang Weiwei Zhang The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Resources Search for more papers by this author Rohit A Panchakshari Rohit A Panchakshari Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Contribution: Resources Search for more papers by this author Dingpeng Yang Dingpeng Yang State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Resources Search for more papers by this author Xiong Ji Xiong Ji The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization Search for more papers by this author Yu Cao Yu Cao orcid.org/0000-0003-1409-2068 Shanghai Institute of Precision Medicine, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Contribution: Resources, Supervision Search for more papers by this author Frederick W Alt Frederick W Alt orcid.org/0000-0002-0583-1271 Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Contribution: Resources, Supervision Search for more papers by this author Fei-Long Meng Corresponding Author Fei-Long Meng [email protected] orcid.org/0000-0003-0333-6872 State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jiazhi Hu Corresponding Author Jiazhi Hu [email protected] orcid.org/0000-0002-6345-0039 The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Xia Xie Xia Xie orcid.org/0000-0002-0423-976X State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Tingting Gan Tingting Gan orcid.org/0000-0002-4679-9212 The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization, Formal analysis, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Bing Rao Bing Rao orcid.org/0000-0001-9895-4133 State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Formal analysis, ​Investigation, Writing - review & editing Search for more papers by this author Weiwei Zhang Weiwei Zhang The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Resources Search for more papers by this author Rohit A Panchakshari Rohit A Panchakshari Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Contribution: Resources Search for more papers by this author Dingpeng Yang Dingpeng Yang State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Resources Search for more papers by this author Xiong Ji Xiong Ji The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization Search for more papers by this author Yu Cao Yu Cao orcid.org/0000-0003-1409-2068 Shanghai Institute of Precision Medicine, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Contribution: Resources, Supervision Search for more papers by this author Frederick W Alt Frederick W Alt orcid.org/0000-0002-0583-1271 Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Contribution: Resources, Supervision Search for more papers by this author Fei-Long Meng Corresponding Author Fei-Long Meng [email protected] orcid.org/0000-0003-0333-6872 State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jiazhi Hu Corresponding Author Jiazhi Hu [email protected] orcid.org/0000-0002-6345-0039 The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Contribution: Conceptualization, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Xia Xie1,†, Tingting Gan2,†, Bing Rao1,†, Weiwei Zhang2, Rohit A Panchakshari3,4, Dingpeng Yang1, Xiong Ji2, Yu Cao5, Frederick W Alt3,4, Fei-Long Meng *,1 and Jiazhi Hu *,2 1State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China 2The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China 3Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA 4Department of Genetics, Harvard Medical School, Boston, MA, USA 5Shanghai Institute of Precision Medicine, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China † These authors contributed equally to this work *Corresponding author. Tel: +86 21 54921620; E-mail: [email protected] *Corresponding author. Tel: +86 10 62744611; E-mail: [email protected] The EMBO Journal (2022)41:e109324https://doi.org/10.15252/embj.2021109324 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In activated B cells, activation-induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C-terminal peptide. In immunodeficient-patient cells expressing mutant AID lacking its C-terminus, a catalytically active AID-delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID-delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID-delC proteins form condensates both in vivo and in vitro, dependent on its N-terminus and on a surface arginine-rich patch. Co-expression of AID-delC and wild-type AID leads to an unbalanced nuclear AID-delC/AID ratio, with AID-delC proteins able to trap wild-type AID in condensates, resulting in a dominant-negative phenotype that could contribute to immunodeficiency. The co-condensation model of mutant and wild-type proteins could be an alternative explanation for the dominant-negative effect in genetic disorders. Synopsis Immunodeficiency-associated activation-induced deaminase (AID) variants lacking the C-terminus behave as dominant-negative in antibody diversification. This study reveals co-condensation as a basis of this dominant-negative effect, with mutant AID proteins forming condensates and trapping wild-type AID from its substrates. The AID C-terminus is required for its recruitment to genomic antibody switch regions C-terminal-deleted AID protein forms condensates in vitro and in vivo AID condensation causes a faulty distribution of deaminase activity C-terminal-deleted AID co-condensates with wild-type protein, resulting in the dominant-negative effect Introduction Antigen receptor diversification is one of the hallmarks of adaptive immunity. Lymphocytes employ programmed DNA lesions to initiate diversification processes and generate diverse antigen receptor repertoires (Alt et al, 2013). In developing B cells, V(D)J recombination assembles the antigen receptor variable exons to form the primary antibody repertoire. Upon antigen stimulation, naïve B cells experience another two diversification processes, named class switch recombination (CSR) and somatic hypermutation (SHM), to further diversify the antibody repertoire. CSR switches the immunoglobulin (Ig) constant gene to change its effector function, while SHM mutates the variable exons to allow antibody affinity maturation (Alt et al, 2013). The resulting antigen receptor repertoires help to recognize diverse pathogens including infectious viruses. Activation-induced cytidine deaminase (AID), coded by the AICDA gene, initiates both CSR and SHM (Muramatsu et al, 2000) by directly deaminating the cytidines (C) at Ig heavy chain (IgH) switch (S) regions or Ig variable exons, respectively. Besides the on-target Ig loci, AID frequently miss-targets a group of off-target sites associated with intragenic super-enhancers (Meng et al, 2014; Qian et al, 2014), leading to B cell malignancies (Casellas et al, 2016). Thus, the mutator activity of AID is highly regulated in B cells (Yeap & Meng, 2019). AID protein contains a nuclear localization signal (NLS) sequence at the N-terminus and a nuclear export signal (NES) sequence at the C-terminus (Brar et al, 2004; Ito et al, 2004; McBride et al, 2004). Wild-type AID protein is mostly located in the cytoplasm, while the NES-mutated AID mutants prefer to accumulate in the nucleus (Ito et al, 2004; McBride et al, 2004). Nuclear accumulation of AID mutator protein potentially possesses a great threat to genome integrity. The C-terminal NES sequence is not required for its deaminase activity in vitro (Mu et al, 2012; Qiao et al, 2017), and ectopically overexpressed NES-mutated AID protein can efficiently deaminate E. coli genomic DNA (Barreto et al, 2003; Bransteitter et al, 2004), generate substantial mutations in chicken B-lineage DT40 cells (Barreto et al, 2003; Geisberger et al, 2009) and mouse fibroblasts (Ta et al, 2003). Surprisingly, a group of AID C-terminal deletion (AIDΔC) alleles, which lack the C-terminal NES, were reported in immunodeficient Hyper-IgM syndrome type 2 (HIGM2) patients, including R190X, V186X, etc. (Durandy et al, 2007). Similarly, in mouse B cells, the nuclear-retention AIDΔC mutants fail to support CSR in ex vivo cytokine-activated B cells (Barreto et al, 2003; Ta et al, 2003; Geisberger et al, 2009; Zahn et al, 2014). The defective CSR in AIDΔC expression B cells leads to the hypothesis that the AID C-terminus has an NES-independent role(s), for example, replacement of the endogenous NES sequence with other NESs can restore the nuclear export but not CSR (Geisberger et al, 2009). The C-terminus could affect protein stability and/or interact with unknown factors (Geisberger et al, 2009; Ellyard et al, 2011). In this context, C-terminus-dependent interactions with polyA RNA and/or hnRNP were proposed (Nonaka et al, 2009; Hu et al, 2015; Mondal et al, 2016). Similarly, AID C-terminus was proposed to be involved in DNA repair and recombination in CSR, for example, affecting recombinant end pairing (Sabouri et al, 2014), recruiting UNG and Msh2 protein to antibody gene (Ranjit et al, 2011), or affecting the functions of DNA damage response factors (Zahn et al, 2014). How this link between AID and downstream DNA repair factors contributes to physiological CSR is not clear, as CSR is processed in a two-step manner (Zarrin et al, 2007), that is, AID initiates DNA lesions first and the general DNA repair factors finally join the breaks. Among the deleterious AICDA mutations in HIGM2 (Revy et al, 2000), most of the HIGM2 patient-derived AICDA mutants are autosomal recessive mutants (Ta et al, 2003) and highly correlated to AID protein stability or catalytic activity (Qiao et al, 2017). However, AIDΔC alleles were reported to be autosomal dominant mutants (Durandy et al, 2007). Dominant mutations inhibit the functions of wild-type gene products through the formation of nonfunctional self-assembled oligomers or competition of limited substrates/cofactors (Herskowitz, 1987). AIDΔC mutants cannot be simply grouped into these two categories. In the current study, we find the AIDΔC mutants fail to target antibody genes and frequently infiltrate into nuclear membraneless organelles through their intrinsic sequence features. Wild-type AID proteins, which dynamically shuttle between nucleus and cytoplasm, are trapped within these subnuclear structures in presence of AIDΔC protein, resulting in the dominant-negative effect. Results Guided AIDΔC can support high levels of CSR Pathogenic mutant AIDR190X with truncation of the C-terminal 9 amino acids (aa) failed to support efficient CSR in CSR-activated B cells (Appendix Fig S1A) and CH12F3 B cell line (Appendix Fig S1B), consistent with previous reports (Barreto et al, 2003; Ta et al, 2003; Geisberger et al, 2009; Zahn et al, 2014). The defective CSR in AIDR190X-complemented B cells cannot be attributed to nuclear protein levels (Appendix Fig S1C and D), or its deamination activity (Appendix Fig S1E and F). To examine the IgH breakage and rearrangements in activated B cells expressing different AID variants, we applied the high-throughput genome-wide translocation sequencing (HTGTS) (Hu et al, 2016), which presents both "bait" Sμ DSBs and CSR junctions. We applied HTGTS with the same numbers of B cells and detected marginal levels of CSR junctions from B cells complemented with AIDR190X and AIDcry with low CSR levels (Appendix Fig S2A). For the bait Sμ DSBs, wild-type AID generated a great number of broken ends, which can be visualized as spikes at RGYW (R: A/G; Y: T/C; W: A/T) motifs (Appendix Fig S2B). However, no such specific enrichments at RGYW motifs were observed in B cells complemented with AIDΔC mutants (Appendix Fig S2B). Therefore, the catalytic-active AIDΔC protein induces fewer DNA breaks at the IgH locus. To test whether reduced AID recruitment at IgH locus or insufficient processing of AID deamination products to breaks leads to the observed phenotypes, we constructed a synthetically guided AIDΔC by two independent approaches and examined the CSR levels in the respective cells. In the first, we fused AID variants with the CRISPR/Cas9 modules (Fig 1A). When the AIDΔC protein was fused to nuclease-dead Streptococcus pyogenes Cas9 (dSpCas9) or MS2 coat protein (MCP), its deamination activity could be directed to a specific genomic locus with a single-guide RNA (sgRNA) or sgRNA with MS2 hairpins (sgRNAMS2) to generate DNA mutations (Ma et al, 2016; Liu et al, 2018). We thus tested whether guided AIDΔC protein could support CSR in B cells. Given the repetitive nature of S regions, we designed sgRNA targeting Sμ and Sα multiple times (Sμ: 44 times; Sα: 10 times; allowing 1 bp mismatch in a 12 bp-long seed sequence proximal to the NRG PAM, Appendix Table S2, Appendix Fig S3A). The multiple recognition sites in S regions ensured the efficient Cas9-initiated CSR when wild-type SpCas9 was used (Appendix Fig S3A). Therefore, we fused AID or AIDΔC mutants to MCP and introduced different MCP-AID variants together with the dSpCas9 and sgRNAMS2 (a system dubbed CRISPR-guided AID, Fig 1A) into AID-deficient CH12F3 cells. Different MCP-AID variants were expressed at comparable levels, with relatively higher MCP-AID levels than others (Appendix Fig S3B). The CRISPR-guided wild-type AID could induce substantial CSR from IgM to IgA, while higher efficiencies were observed by using AIDcry and AIDR190X (Fig 1A). To visualize the CRISPR-guided AID-generated breaks at S regions, we performed a quantitative version of HTGTS referred to as PEM-seq (primer-extension-mediated sequencing) (Yin et al, 2019), which can quantitatively reveal end-joining initiated by CRISPR/Cas9 tools. We employed Staphylococcus aureus Cas9 (SaCas9) and an sgRNA targeting the Iγ3 region to induce "bait" DSBs for preparing PEM-seq libraries. Substantial translocation junctions were captured between Iγ3 bait and Sμ or Sα in PEM-seq libraries from CRISPR-guided AID variants expressing AID-deficient CH12F3 cells, and the junction numbers at both S regions (Fig 1B) were well-correlated with the CSR levels (Fig 1A). Figure 1. AID C-terminal tail is required for its targeting to IgH substrates CRISPR-guided AIDΔC protein could support CSR in Aicda−/− CH12F3 cells. Left, CRISPR-guided AID is schematically illustrated with each component listed. Right, CRISPR-guided AID-initiated CSR levels to IgA. Blue arrows represent multiple CRISPR recognition sites. The number in the parentheses indicates CRISPR recognition sites in each S region. Three biological replicates are plotted by dots and mean with standard deviation (SD). CRISPR-guided AID-generated breaks at the S region in Aicda−/− CH12F3 cells were quantitatively detected by PEM-seq. Top, schematic illustration shows the bait SaCas9 site at Iγ3 region and CRISPR-guided AID variants targeting sites at S regions. Bottom, percentages of translocation junctions between Iγ3 and Sμ or Sα regions are plotted as mean with SD of three biological replicates in the bar plot. AID-AIDE58Q dimer proteins were expressed in AID-deficient CSR-activated B cells, AID dimer is schematically illustrated on top and CSR levels to IgG1 are shown. Three biological replicates are plotted by dots and mean with SD is shown. CSR levels to IgA in Aicda−/− CH12F3-miniS cells with indicated AID variants or CRISPR-guided AID variants. Levels of CSR to IgA are plotted as mean with SD in the bar plot (six biological replicates for EV, AID, AIDR190X, and three biological replicates for other samples). Mutation profiles of miniS region in Ung−/−Msh2−/−Aicda−/− miniS cell lines complemented with indicated AID. Mutation frequency at each nucleotide along the 756-bp miniS region is plotted as a bar graph with green error bars, representing mean with standard error of the mean (SEM) of three biological replicates. Positions of AID-preferred AGCT and other RGYW motifs are marked with orange and yellow respectively under each plot. Mutation profiles of miniS region in Ung−/−Msh2−/−Aicda−/− miniS cell lines expressing CRISPR-guided AID variants. Mutation frequency at each nucleotide is plotted as mean with SEM of three biological replicates in the bar graph with green error bar. Plots are labeled as in (B). The sgRNA targeting position is indicated with blue lines at the bottom. Data information: For panels A, B, and C, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. Unpaired two-tailed Student's t-test was performed for panel D. For all panels, ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P > 0.05. Source data are available online for this figure. Source Data for Figure 1 [embj2021109324-sup-0002-SDataFig1.zip] Download figure Download PowerPoint In the second approach, AIDΔC was synthetically recruited to endogenous IgH locus in B cells through a dimerization method. Among the AID/APOBEC family members, several of them contain two tandem domains, for example, APOBEC3B, APOBEC3F, and APOBEC3G (Harris & Dudley, 2015). A similar dimer configuration has been applied to access the function of AID mutants by fusing AID mutants with the full-length catalytic-dead AIDE58Q (Methot et al, 2018). Thus, we generated a panel of synthetic AIDΔC-AIDE58Q dimer proteins (Fig 1C), in which setting the full-length AIDE58Q could recruit AIDΔC to the IgH locus. Similar to the CRISPR-guided AID, the AIDΔC-AIDE58Q dimer proteins supported substantial levels of CSR (Fig 1C), though the levels of dimer proteins were lower than those of the monomers (Appendix Fig S3C). Therefore, with two different approaches to synthetic recruitment, synthetic-recruited AIDΔC can support substantial levels of CSR in vivo. Guided AIDΔC can support deamination-initiated mutagenesis in an S region To further test the AID targeting in S regions, we generated a CH12F3-miniS cell line which allows the assessment of the mutation spectrum at an intact mini-switch region. In the AID-deficient CH12F3-miniS cells, the endogenous Sα was replaced with a 756-bp core Sμ sequence via a recombinase-mediated cassette exchange approach (Han et al, 2011) (Appendix Fig S3D and E). Both wild-type AID and the MCP-tagged wild-type AID could support substantial levels of CSR in the AID-deficient CH12F3-miniS cells (Fig 1D, columns 2 and 6 from left), while the latter showed a relatively lower CSR level. AIDΔC failed to support efficient CSR in CH12F3-miniS cells (Fig 1D, columns 3,4 and 7,8 from left; Appendix Fig S3F). However, in the CRISPR-guided approach, both AID and AIDΔC supported substantial levels of CSR under the same treatments at comparable protein levels (Fig 1D and Appendix Fig S3G), similar to the findings in the parental CH12F3 cells (Fig 1A). CRISPR-guided AIDΔC induced a higher CSR level to IgA than guided AID in CH12F3-miniS cells, reaching more than 10%. The CH12F3-miniS cells allow us to sequence through the whole miniS region to check for AID footprints. To trace AID deamination sites, we knocked out Ung and Msh2 in CH12F3-miniS cells (Fig 1E and Appendix Fig S3E), in which genetic background the bona fide AID deamination events are processed during DNA replication and can be visualized by C>T transition mutations (Rada et al, 2004). We found that the mutation frequency at the miniS region was greatly decreased in AIDR190X-expressing cells compared to that in wild-type AID-expressing cells (Fig 1E), indicating a decreased targeting of AIDR190X. In the same context, CRISPR-guided AIDΔC efficiently generated C>T transition mutations around the CRISPR recognition sites (Fig 1F), and the mutation frequencies of different AID variants were consistent with the CSR levels (Fig 1D, last three columns from left). Collectively, the results demonstrate that AID C-terminus is crucial for its recruitment to the S regions, and the AIDΔC deamination products can be efficiently channeled into recombination events. AIDΔC protein has the tendency of condensation in cells To investigate the mechanism underlying the defective recruitment of AIDΔC, we ectopically expressed AID and mutants in CH12F3 cells. Under the current experimental condition, AID expression levels measured by the mRNA quantity were similar to those of the endogenous AID expression in CH12F3 cells stimulated by cytokines (Appendix Fig S4A). AID-GFP protein was dominantly located in the cytoplasm, and partially retained in the nucleus upon CRM1 inhibitor (CRM1i) treatment (Appendix Fig S4B), as previously reported (Brar et al, 2004; McBride et al, 2004; Patenaude et al, 2009). AIDΔC-GFP proteins, including AIDR190X-GFP and AIDcry-GFP, were constitutively retained in the nuclei (Appendix Fig S4C), with a relative enrichment in the nucleoli marked by Fibrillarin (FBL) (Appendix Fig S4B). In addition, AIDΔC-GFP puncta were occasionally observed outside the nucleolus (Appendix Fig S4B, arrowhead). When increasing the protein levels by overexpressing GFP-fused AID variants in the U2OS cells, we found that AIDR190X puncta can also co-localize with nucleolus and other nuclear membraneless organelles, such as Cajal bodies, gems, nuclear speckles, or peri-nucleolar compartments (Appendix Fig S4D), as previous observations in Hela cells (Hu et al, 2013, 2014). To test whether the AIDΔC puncta co-localize with the IgH targets in B cells, we visualized the endogenous IgH locus by using the CRISPR-Sirius system in live CH12F3 cells, in which assay the IgH Sμ region was labeled with the multiple-targeting sgRNA (Fig 2A and Appendix Table S2). The CRISPR-Sirius assay consistently detected two IgH loci in CH12F3 cells, and none co-localized with AIDΔC puncta (Fig 2A). Therefore, these results indicate that the frequent infiltration of AIDΔC proteins into nuclear compartments might affect their targeting of the physiological IgH substrate. Figure 2. The condensation of AID-delC reduces its activity in CSR Representative images showing the localization of AID (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda−/− CH12F3 cells. The IgH loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH locus visualization is shown on top. Scale bar, 5 µm. OptoDroplet formation in CH12F3 cells. Schematic illustration of light-induced optoDroplet system is depicted on top. AID variants were tagged to the N-terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time. Representative images after the third blue light activation in Aicda−/− CH12F3 cells with the indicated AID variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP-FBL, respectively. Scale bar, 5 µm. Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells. CRM1 inhibitor (CRM1i) was used for selective trapping of AID in nuclei. Scale bar, 5 µm. The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell, and more than 20 cells represent two biological replicate samples were counted. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****P < 0.0001; *P < 0.05; ns, P > 0.05. Intensity recovery lines for o