Quick ligation kit

Four single‐guide RNAs (sgRNAs), sgRNA1/2 and sgRNA3/4 (see Supporting Information Table S1), were cloned into a pSpCas9(BB)‐2A‐green fluorescent protein (GFP) (PX458) plasmid containing the SpCas9 gene with 2A‐enhanced green fluorescent protein (EGFP) and the backbone of the sgRNA (Addgene plasmid #48138). The sequences for sgRNA1/2 have been used and reported previously 23 The sgRNA3/4 were designed in our lab to target exon 23 using an sgRNA CRISPR design tool (http://crispr.mit.edu). Cloning of sgRNA was done according to the protocol of Dr. Feng Zhang's lab at the Massachusetts Institute of Technology. In brief, complimentary oligos that contained the appropriate sgRNA sequences and BbsI restriction sites were obtained from Integrated DNA Technologies and annealed to form double‐stranded DNA fragments. The resulting fragments and backbone plasmids were digested with BbsI and ligated using a Quick Ligation Kit (New England Biolabs, Ipswich, MA).

Genomic DNA was purified individually from human myeloid and lymphoid cells engrafted in mice. LAM-PCR analysis was performed as described previously.^{17 (link)} Vector LTR junction sequences were amplified by linear PCR using 300 ng of genomic DNA, vector LTR–specific 5′ biotinylated primer, 5′-AGAACCTTGTGTCTCTCATCCC-3′, and PCR master mix. DNA was denatured at 94 °C for 5 minutes, followed by 50 cycles of amplification (94 °C for 45 seconds, 60 °C for 45 seconds, and 72 °C for 60 seconds), and a final extension at 72 °C for 10 minutes. Biotinylated PCR products were bound to streptavidin-conjugated magnetic beads overnight with mild shaking. After washing, double-stranded DNA was synthesized using a random hexamer (Invitrogen) and DNA polymerase I large fragment (NEB, Ipswich, MA). DNA was digested with MluCI, MseI, and NlaIII (NEB) and ligated to restriction enzyme-specific double-stranded linkers using the Quick Ligation Kit (NEB). DNA was denatured with 0.1 N NaOH and was PCR-amplified using LTR- and linker-specific primers (first nested PCR primer of LTR, 5′-ACCTCCTTCCCTGTAATACTC-3′, and primer of linker, 5′-GCACTCGTGCTCGACTGATAC-3′; second nested PCR primer of LTR, 5′-CCTGGTTTCTAGTGGCATTC-3′ and primer of linker, 5′-CCGTCGTATCGTAGCACAG-3′). A schematic diagram shows the LAM-PCR analysis (see Supplementary Figure S4).