Non-homologous end joining (NHEJ) is the first-responder pathway at Cas9-induced DSBs, ligating broken ends through Ku70/Ku80 recruitment and DNA-PKcs-mediated synapsis to produce small insertions and deletions under 50 base pairs (PMID: 36269834). NHEJ-mediated repair typically saturates within 24 hours of Cas9 delivery. However, when NHEJ fails to resolve the break promptly, 5'-to-3' end resection by the MRN complex (MRE11/RAD50/NBS1) and CtIP generates single-stranded DNA overhangs that expose microhomology tracts flanking the cut site. These resected ends are channeled into microhomology-mediated end joining (MMEJ), which aligns short homologous sequences (5 to 25 bp) across the break junction and deletes the intervening DNA, producing kilobase-scale deletions that continue to accumulate for up to 72 hours post-editing (PMID: 36269834, 41736887). The guide RNA sequence determines the balance between these outcomes: the R-02 gRNA predominantly generates a 9 bp in-frame deletion via a local MMEJ microhomology, yielding lower rates of large deletions, whereas the R-66S gRNA produces a more diverse, frameshift-skewed indel spectrum with higher large-deletion frequency (PMID: 41736887, 36269834).

Pharmacological inhibition of NHEJ using the DNA-PKcs inhibitor M3814 was designed to shift repair toward HDR and improve therapeutic knock-in rates, but paradoxically increases the formation of large genomic modifications and loss of allele events by extending the window of unresolved DSBs available for MMEJ processing and chromosomal missegregation (PMID: 41736887). The REC3 domain of Cas9 functions as a conformational checkpoint sensor that governs the allosteric transition of the HNH nuclease domain to its catalytically active state upon RNA/DNA heteroduplex recognition, and variations in this checkpoint stringency across engineered high-fidelity Cas9 variants influence the kinetics of DSB formation and consequently the repair pathway balance at the HBB locus (PMID: 28931002).

When DSBs persist unrepaired through S phase and into mitosis, the Cas9 cut site divides chromosome 11 into a centric fragment (retaining the centromere) and an acentric fragment (lacking centromeric attachment). The acentric fragment, unable to attach to the mitotic spindle, missegregates during anaphase and is partitioned into a micronucleus (PMID: 34615869). Within micronuclei, the encapsulated chromosome undergoes severely defective DNA replication, detectable as reduced EdU incorporation, causing progressive degradation and permanent loss of the genomic material distal to the cut site. Concurrently, fusion of the broken centric ends of sister chromatids generates dicentric chromosomes that form anaphase bridges during subsequent divisions; resolution of these bridges can result in co-segregation of both chromosome 11 homologs to a single daughter cell, leaving the other daughter with whole-chromosome monosomy (PMID: 34615869). Interhomologue recombination (IHR) adds a further layer of complexity: Cas9-induced DSBs at HBB can be repaired using the highly homologous HBD gene as an endogenous template, producing gene conversion events that correct the sickle mutation but simultaneously generate 7.4 kb deletions spanning the HBB-to-HBD interval, and concurrent off-target DSBs at the OT18 site on chromosome 11q can produce 54 Mb pericentric deletions or inversions (PMID: 31147717, 37069266, 25894090).