Pet28a

Human SPI1 cDNA (816 bp, Shanghai GeneChem Co., Ltd, China) were inserted into CV186 lentivirus vector (Genechem Co., Ltd). Human SPIB cDNA (789 bp) construct was provided by Dr. Zhe Liu.⁴⁷ Their truncated fragments obtained using primer sets (Table S5) were inserted into pCMV‐N‐Myc, pCMV‐3Tag‐1C, pGEX‐6P‐1 or pET‐28a (Addgene, Watertown, MA). The shRNAs for SPI1 or SPIB were established by inserting oligonucleotides (Table S6) into GV298 vector (Shanghai GeneChem Co., Ltd), while small interfering RNAs (siRNAs) were synthesized (Table S6).

The human full-length 14-3-3σ cDNA was subcloned into the prokaryotic expression vector pET28a (+) (Addgene, Cambridge, MA) for expressing a fusion protein tagged with histidine (His). The N-terminal His₆-tagged 14-3-3σ vectors were transformed into BL-21 (DE3) bacteria (Yeastern Biotech Co., Taipei, Taiwan) and grown at 37°C in LB medium with 50μg/mL kanamycin followed by induction with 1 mM isopropyl-β-D-thiogalactoside (IPTG). His-tagged 14-3-3σ was amplified and purified by using a Ni-Ten resin according to the manufacturer's instructions (Macherey-Nagel GmbH & Co. KG, Düren, Germany).

All plasmids were transfected with Lipofectamine 2000 (Life Technologies-Invitrogen, USA) according to the manufacturer’s instructions. H1.2, H1.3 and H1.4 cDNAs were amplified and cloned into the p3× FLAG-CMV-10 vector (Addgene, USA). Full-length H1.2 and various fragments (N-terminal domain, 1–35 aa; globular domain, 36–112 aa; C-terminal domain, 113–213 aa; ΔC1, 1–179 aa; ΔC2, 1–112+180–213 aa; ΔC, 1–112 aa) were cloned into the pEGFP-C2, pGEX-4T3 or pET28a vectors (Addgene, USA). The full-length FLAG-ATM expression construct was purchased from Addgene, USA. GST-ATM fragments (F1, 1–247 aa; F2, 250–522 aa; F3, 523–769 aa; F4, 722–1102 aa; F5, 1098–1371 aa; F6, 1245–1435 aa; F7, 1239–1770 aa; F8, 1764–2138 aa; F9, 2141–2428 aa; F10, 2427–2841 aa; F11, 2842–3056 aa; F12, 2682–3012 aa) were provided by Dr. Fabrizio d’Adda di Fagagna (FIRC Institute of Molecular Oncology Foundation, Italy). Site-specific mutations of H1.2 (T126/146/165A, T126/146/165E, E115A, S173A, S188A) were generated using a site-directed mutagenesis kit (Vazyme, China).

HT-29 and THP-1 cells used in this study were purchased from the American Type Culture Collection (ATCC, UDA). HT-29 cells were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum (FBS). THP-1 cells were grown in RPMI 1640, supplemented with 10% FBS and 0.05 mM 2-mercapto ethanol. Salmonella Typhi Ty2, a gift from J. Parkhill (Sanger Institute, Hinxton, UK) was grown in Hektoen enteric agar (BD Difco). Escherichia coli strain BL21, a kind gift from Dr. Rupak k. Bhadra (Indian Institute of Chemical Biology) was maintained in Luria–Bertani agar at 37 °C. Liquid cultures of the bacterial strains were grown in Luria-Bertani broth (BD Difco). pET-28a was purchased from Addgene, USA.

The VirD2 open reading frame was PCR amplified from the total DNA isolated from Agrobacterium tumefacien strain GV3101 and cloned into the pENTER-D-Topo vector, and the sequence was confirmed by Sanger sequencing. To express Cas9-VirD2 or VirD2-Cas9 under the T7 promoter, the VirD2 open reading frame was fused in-frame to the N or C terminus of Cas9 in pET28a (Addgene plasmid #53261) following the Gibson assembly protocol. For in planta expression, the plant codon-optimized sequence of VirD2 was custom synthesized and cloned in-frame to either the N or C terminus of Cas9 in pRGE32 and pRGEB32 (Addgene plasmid #63142) following the Gibson assembly protocol. sgRNAs as a primer dimer were first cloned into the entry clone under the U3 promoter by Golden Gate cloning with BsaI. The whole cassette, U3::sgRNA, was PCR amplified and cloned into the HindIII and SbfI sites in the pRGEB-Cas9-VirD2 and pRGEB-VirD2-Cas9 vectors. The sequences of all primers used in PCR amplification are provided in Supplementary Table 3.