Table 2.

Guide-Seq in HEK293-Cas9 Cells Offers the Widest Identification Potential for Off-Target Risks in CD34⁺ HSPCs

RAG2 Sites Detected by rhAmpSeq in HSPCs (Editing > 0.1%)		RAG2 Sites Detected by GUIDE-seq (Using Different Cell Types and gRNA Formulations)
Site	Genomic Position	Alt-R XT 2-Part RNA HEK293-Cas9	Alt-R sgRNA HEK293-Cas9	Alt-R XT 2-Part RNA HEK293 RNP	Alt-R sgRNA HEK293 RNP	Alt-R sgRNA HSPCs RNP
2–on	Chr11:36594108	+	+	+	+	+
1	Chr3:182671721	+	+	+	+	+
4	Chr4:102437906	+	+	+	+	+
5	Chr3:171315260	+	+	+	+	−
7	Chr13:85131231	+	+	+	+	−
9	Chr1:86729397	+	+	−	−	−
12	Chr3:136434089	+	−	−	−	−
13	Chr6:154714216	+	+	−	−	+

RAG1 Sites Detected by rhAmpSeq in HSPCs (Editing > 0.1%)		RAG1 Sites Detected by GUIDE-seq (Using Different Cell Types and gRNA Formulations)
Site	Genomic Position	Alt-R XT 2-Part RNA HEK293-Cas9	Alt-R sgRNA HEK293-Cas9	Alt-R XT 2-Part RNA HEK293 RNP	Alt-R sgRNA HEK293 RNP	Alt-R sgRNA HSPCs RNP
1–on	Chr11:36573321	+	+	+	+	+
2	Chr1:16415506	+	+	+	+	+
4	Chr11:57348913	+	+	+	−	−
5	Chr3:140708416	+	+	−	+	−
8	Chr1:34144141	+	+	+	+	+
19	Chr19:4360240	+	+	+	+	+
35	Chr12:90948235	+	+	+	+	+
37	Chr6: 26286921	+	+	−	−	−

This table presents a comparison of the GUIDE-seq identification potential under different experimental conditions for confirmed off-target sites that had the rhAmpSeq activity level above 0.1% in CD34⁺ HSPCs. Chr, chromosome.