Table 2.

Methods used or developed to detect SVs in edited human cells

Method	Description	Advantages	Limitations	References
Fluorescent in situ hybridization (FISH)	Chromosome structure is analyzed using defined fluorescent DNA probes observed in condensed chromosomes	Can detect large chromosomal aberrations, such as truncations and translocations	Has low-resolution and sensitivity. Is time-consuming when scaled. Requires live cells	(Boutin et al. 2021; Cullot et al. 2019; Leibowitz et al. 2021; Przewrocka et al. 2020; Rayner et al. 2019)
Array-comparative genomic hybridization (aCGH)	Genome wide DNA copy number analysis is performed based on the relative intensity of complementary fluorescent probes between sample and control DNA	Can detect SVs that result in copy-number gain or loss	Detection threshold not suitable for pooled DNA. Cannot detect the location of copies or copy number neutral SVs	(Boutin et al. 2021; Cullot et al. 2019; Przewrocka et al. 2020)
Quantitative PCR (qPCR) and quantitative genotyping PCR (qgPCR)	A modified PCR which can quantify the allele copy number of a target site post editing	Can detect SVs that result in copy-number gain or loss of the specific amplification site	Detection threshold not suitable for pooled DNA. Cannot detect the location of copies or copy number neural SVs	(Boutin et al. 2021; Simkin et al. 2022; Weisheit et al. 2020, 2021)
Single-nucleotide polymorphism (SNP) genotyping	Heterozygous SNP allelic ratios are analyzed by either targeted amplification and sequencing or SNP-array methods	Can detect chromosomal aberrations or SVs which result in LOH of SNP sites e.g., deletions and truncations	Detection threshold not suitable for pooled DNA. Cannot distinguish between deletions and CN-LOH	(Alanis-Lobato et al. 2021; Leibowitz et al. 2021; Przewrocka et al. 2020; Simkin et al. 2022; Weisheit et al. 2020; Zuccaro et al. 2020)
Targeted short- and long-amplicon sequencing	Targeted PCR amplification followed by NGS	Can detect SVs housed completely within the amplicon	Limited to SVs housed completely within the amplicon. Allele quantification is prone to PCR and sequencing bias	(Kosicki et al. 2018; Quan et al. 2022; Simkin et al. 2022; Wen et al. 2021; Yoo et al. 2022)
Individual DNA molecule sequencing (IDMseq)	Individual DNA molecules are labeled with UMIs followed by the generation of short- or long amplicons and NGS	Detect and quantify SVs housed completely within the amplicon	Limited to SVs housed completely within the amplicon	(Bi et al. 2020)
Linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS)	Linear amplification across a target DSB site using a biotinylated primer. Amplicons are enriched by streptavidin selection and are further amplified after the ligation of an adapter. Amplicons not containing SVs are ablated using a rare-cutting restriction enzyme and the remaining amplicons are sequenced by NGS	Can detect SVs where the break point is proximal to the primer site	Cannot detect SVs where the primer site has been ablated. Allele quantification is prone to PCR and sequencing bias	(Hu et al. 2016)
Primer extension-mediated sequencing (PEM-seq)	Primer extension across a target DSB site using a biotinylated primer. Amplicons are enriched by streptavidin followed by the ligation of a barcoded adapter. Nested amplification is performed before sequencing by NGS	Can detect and quantify SVs where the break point is proximal to the primer site	Cannot detect SVs where the target primer site has been ablated	(Liu et al. 2021; Wu et al. 2022; Xin et al. 2022; Yin et al. 2019; Yin et al. 2022; Zhang et al. 2021)
Chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-seq)	Pooled DNA is fragmented and linkers are ligated. Fragments are then amplified by PCR using ‘bait’ and linker primers so that fragments which do not contain SVs are ablated by the included ‘decoy primers’. The amplicons are then sequenced by NGS	Can detect SVs where the break point is proximal to the primer site	Cannot detect SVs where the target primer site has been ablated. Quantification requires ddPCR calibration. Does not quantify DNA without SVs	(Turchiano et al. 2021)
Whole-genome sequencing	Unbiased next-generation sequencing of whole genomic DNA	Can detect all types of variants including SNPs, INDELs and SVs at all sites	Detection threshold not suitable for pooled DNA	(Alanis-Lobato et al. 2021; Schmidt et al. 2023; Simkin et al. 2022)
Xdrop	Encapsulated PCR of fragmented HMW DNA with primers for a 100–200 bp target, distal (< 5 kb) to the break point. Droplets containing the fragments of interest can be identified with an intercalating fluorescent dye and sorted by flow cytometer droplet sorting. Enriched HMW DNA is then amplified by MDA followed by NGS	Can detect SVs where the break point is within the enriched HMW DNA	Cannot detect SVs where the target primer site(s) has been ablated	(Blondal et al. 2021; Geng et al. 2022)
Prime editor-assisted off-target characterization (PEAC-seq)	A Cas9, M-MLV reverse transcriptase fusion protein is used to generate and tag DSBs at off-target sites. Bulk DNA is fragmented by Tn5 tagmentation, which introduces UMI and adapter sequences. For identification of translocations, nested PCR is performed with target-specific forward and Tn5-specific reverse primers. Amplicons are then subjected to NGS	Can detect and quantify SVs where the break point is proximal to the primer site. Can also be used to identify candidate off-target sites	Low or variable efficiency of DSB and tag process. Acts as a reporter method. Cannot be applied to bulk DNA that was edited using alternative methods	(Yu et al. 2022)

ddPCR digital droplet PCR, LOH loss of heterozygosity, CN-LOH copy-neutral LOH, HMW high molecular weight, MDA multiple displacement amplification, M-MLA moloney murine leukemia virus, NGS next-generation sequencing, SNP single-nucleotide polymorphism, UMI unique molecular identifier