pX330-U6-Chimeric_BB-CBh-hSpCas9

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Genomic deletion of glycoTs in leukocytes
Target sites on human COSMC, UGCG and MGAT1 were determined using the CRISPR Design tool by minimizing exonic off-target effects41. Target sequences were cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene, Plasmid# 42230) after BbsI digestion. COSMC-KO ([O]−cells), MGAT1-KO ([N]−cells) and UGCG-KO ([G]−cells) single KO cell lines were created by electroporating WT HL-60s with vectors containing target sequences (Fig. 2) against the respective genes. COSMC/UGCG-KO dual KOs (i.e. ([OG]−cells) were made by co-transfection of HL-60 with an equimolar plasmid mixture containing COSMC and UGCG targeting plasmids. Dual KO COSMC/MGAT1-KO ([ON]−) and MGAT1/UGCG-KO ([NG]−), and triple KO MGAT1/COSMC/UGCG-KO ([NOG]−) and UGCG/COSMC/MGAT1-KO ([GON]−) cell lines were produced by transfecting [N]−, [G]−, [OG]− or [ON]− cells, respectively, with a vector targeting either COSMC, MGAT1 or UGCG. In all cases, HL60 growth media was changed to RPMI-1640 with 10% FBS at least 48 h prior to electroporation using the Neon Transfection System (Invitrogen). Cells were electroporated according to the manufacturer’s recommendation. After electroporation, cells were cultured in RPMI 1640 with 10% FBS without antibiotics overnight and then supplemented with 50/50 (v/v) mixture of fresh and HL-60 cell-culture conditioned RPMI 1640 (conditioned media). Approximately 2–3 weeks thereafter, both single-cell and bulk-cell FACS sorting (BD FACSAria) was performed using high VVA-FITC binding to select for the COSMC-KOs and low L-PHA-FITC staining to detect Mgat1 deletion. Conditioned media was necessary for single cell cultures to maintain cell viability. After scale up, culture media was changed back to IMDM with 10% FBS for all functional studies. To characterize the KOs, the region surrounding the CRISPR target site was PCR amplified from genomic DNA with primers in Supplemental Table S1 and sequenced. Clones were checked for exonic off-target mutations predicted by the CRISPR Design tool. Off-target sites and PCR primers for COSMC and UGCG gRNA are listed in Supplementary Tables S2 and S3, respectively. MGAT1 gRNA lacked any predicted exonic off-targets. Off-target editing was absent in all clones characterized in this manuscript (Supp. Tables S2, S3).



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Manufacturer protocol

Download the product protocol from Addgene for pX330-U6-Chimeric_BB-CBh-hSpCas9 below.

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