Sickle cell disease (SCD) is a devastating chronic illness marked by severe pain, end organ damage and early mortality (1, 2). It affects ~100,000 Americans and millions more worldwide (3, 4), but treatment options for SCD remain very limited. Pharmacological therapy with hydroxyurea or chronic blood transfusions at best modulates the disease severity but does not cure patients (5). Currently, the only curative therapy for sickle cell disease (SCD) outside of a limited clinical trial is a hematopoietic stem cell transplant (HSCT), typically from a matched related donor, which is available to only ~15% of patients (6, 7). Morbidity and mortality from HSCT increases significantly when using matched unrelated donors (8), or haploidentical donors (9). A recent prospective study of unrelated donor HSCT in SCD concluded that, without modifications to existing regimens, this therapy is not safe for widespread adoption (10). With the advancement of CRISPR/Cas9 technology, there are several possible gene editing strategies to ameliorate SCD: (i) correction of the causative A-T point mutation in β-globin (HBB)(11-14), (ii) induction of fetal hemoglobin (HbF)(15, 16), and (iii) gene addition of a β- globin, γ-globin, or anti-sickling β-globin cassette (17), among which correction of the A-T mutation or producing high enough levels of HbF could be curative. We and others recently demonstrated that, by delivering CRISPR gRNA/Cas9 ribonucleoproteins (RNPs) together with single-stranded oligonucleotide (ssODN) donor templates into SCD patient-derived hematopoietic stem and progenitor cells (SCD HSPCs), up to ~37% of mutant HBB alleles can be gene corrected (12, 14). Injection of gene-edited SCD HSPCs into immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment, with detectable levels of gene correction 16-19 weeks post-transplantation (14). We have shown that by using a high-fidelity Cas9 that maintained the same level of ontarget gene modification, the off-target effects could be significantly reduced (14). However, potential large deletions and insertions at the HBB on-target cut-site, and off-target effects such as chromosomal translocation and inversion in gene-edited SCD HSPCs remain a significant safety concern, since even a very small number of HSCs harboring these detrimental events could clonally expand in vivo and cause a disease such as cancer. Previously, we optimized droplet digital PCR (ddPCR) assay to quantify large deletions and inversions between the R-66 SCD gRNA target site in HBB and a known off-target site (OT18) in gRNA/Cas9 WT RNP-treated SCD HSPCs (14). For high throughput discovery and quantification of such large modifications, we recently developed two next-generation sequencing (NGS) based methods based on short-read high-throughput illumina NGS platform leveraging the high sensitivity and cost-competitiveness of short-read NGS. The first is the LongAmp-Seq (Long-range PCR Amplification based ...