Pmscv ires gfp

NUDT21 codon-optimization was performed based on the NUDT21 full-length cDNA sequence (Ensembl NUDT21-201, ENST00000300291.10) to exclude EcoRI, XhoI and sgNUDT21.336 target sequences and synthesized with terminal 5’ EcoRI and 3’ XhoI sites using gBlock synthesis (IDT) (Supplementary Table 10). NUDT21 sgRNA-resistant (NUDT21^sgRes) was subcloned into Empty vector (EV) pMSCV-IRES-GFP (Addgene #9044) via EcoRI and XhoI sites. Cas9⁺ NALM6 BCP-ALL cells were co-transduced with amphotrophic virus harboring either pMSCV-IRES-GFP;pLRCherryv2.1-sgROSA, pMSCV-NUDT21^sgRes-IRES-GFP;pLRCherryv2.1-sgROSA, pMSCV-IRES-GFP;pLRCherryv2.1-sgNUDT21.336 or pMSCV-NUDT21^sgRes-IRES-GFP;pLRCherryv2.1-sgNUDT21.336 and cultured for six days prior to flow cytometry, then cultured for a further five days. Fitness was assessed by calculating the percentage GFP⁺mCherry⁺ cells at day 6 and day 11 post-transduction.
Full length (FL) CD19 cDNA (Ensembl CD19–202, ENST00000538922.8) including (CD19^FL) or excluding (CD19^ΔUTR) the 3’ UTR sequence was synthesized using gBlock synthesis (IDT) then subcloned into pMSCV-IRES-GFP (Addgene #9044) via Gibson Assembly (New England BioLabs, E2611) (Supplementary Table 10). Cas9⁺ Reh cells were co-transduced with amphotrophic virus harboring either pMSCV-IRES-GFP, pMSCV- CD19^ΔUTR-IRES-GFP or pMSCV- CD19^FL-IRES-GFP. Cells were cultured for 72-hours prior to flow cytometry.

A single FLAG-tag was added to the N-terminus of the cDNAs of yeast ASP1 or zebrafish zASPG. The cDNAs were inserted into a modified pTRIPZ lentiviral vector (Dharmacon) or pMSCV-IRES-GFP (Addgene). Guide RNAs against human glutamine synthetase (sgGLUL-1, sgGLUL-2 and sgGLUL-3) were cloned into pLentiCRISPR V2 vector. For CRISPR rescue experiment, mouse Glul cDNA was cloned into pTURN-hygro retroviral expression vector, and HA tag was added to the C-terminus. Human ASRGL1 cDNA was cloned into pMSCV-puro retroviral expression vector (Clontech). Lentiviral particles were produced in 293T cells by using psPAX2 and pMD.2 packaging plasmids (Addgene). Retroviral particles were produced in 293T cells by using pcleo and pCMV-VSV-G packaging plasmids (gift from Scott Lowe). Cells were infected with viral supernatant in presence of 6 μg/mL of polybrene overnight and subjected to puromycin (2 μg/mL) or hygromycin (200 μg/mL) selection, or GFP sorting.

To construct the TET3-mCherry and Runx3-blue expression plasmids, the open reading frames of TET3 and RUNX3 were amplified by PCR using the primers listed in Supp. Table 1. The PCR products were cloned into the pMSCV-IRES-GFP (86537, addgene, Cambridge, MA) pMSCV-IRES-mCherry (80139, addgene) and pMSCV-IRES-Blue FP (52115, addgene) vectors using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA).