Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5665_Библиотеки_им_академика_М_И_Перельмана

One TaqMan probe is designed to quantify the total number of alleles present in the sample (NHEJ insensitive reference probe, indicated as “Ref” on Figure14.5C) and another TaqMan probe is designed to quantify the number of edited alleles (NHEJ sensitive probe, indicated as “NHEJ” on Figure14.5C). Due to the impre­cise nature of NHEJ, edited sites contain indels of diverse sequence and therefore, it is not feasible to design a probe that can efficiently bind all possible sequences at the edited site. Instead, the “drop out” probe strategy is employed where the NHEJ sensitive probe is specific to the wild‐type version of the modification site and only produces fluorescent signal if DNA is not modified. Therefore, the num­ber of edited alleles is derived from the lack of signal from the NHEJ sensitive probe. The data are presented as a two‐dimensional plot where the wild‐type DNA signal is derived from NHEJ insensitive/NHEJ sensitive double positive droplets and the edited DNA signal is derived from the droplets with a decrease or loss of the signal from the NHEJ sensitive probe and positive signal from the NHEJ insensitive probe.

This ddPCR method was shown to be highly sensitive with a limit of detection (LOD) of 0.2–0.5% indels[69, 70]. The detention of indel frequency was also accu­rate when compared with NGS and flow cytometry with reported differences in as low as 4%[68]. To achieve such high analytical performance, the assay needs to be carefully optimized. This includes fine tuning of PCR conditions and determining probe sequences that provide the highest possible signal/noise ratio. Ideally, the edited site should be sequenced so that the NHEJ sensitive probe is designed accordingly[71]. Miyaoka etal. extended this assay to simultaneously quantify both HDR‐mediated point mutation correction and NHEJ‐mediated insertions and deletions[72, 73].

methods to detect indels introduced by engineered nucleases. EMC uses mismatch‐ leave DNA homoduplexes intact [76] (Figure 14.5D). Firstly, gDNA is isolated from the edited sample and the genomic locus flanking the edited site is amplified using PCR. The PCR product is then denatured and reannealed resulting in DNA heteroduplexes containing mismatches where one DNA strand is wild‐type and another DNA strand contains an indel. This mixture is then treated with a nucle­ase that recognizes and cleaves the dsDNA at the site of mismatch. Lastly, the products of this reaction are resolved using methods such as gel electrophoresis. The approximate indel frequency is then estimated by quantifying the cleaved and intact DNA as represented by bands of different mobility (Figure14.5D). Optimal conditions for heteroduplex formulation between indel‐bearing and wild‐type DNA strands occurs when indel levels are relatively low (around 10%) and wild‐ type amplicons are in excess. Therefore, a simple formula can be applied to quan­tify percent modification (percent modification=fraction of cleaved bands/2). Once modification levels are higher, the formula needs to be corrected to account for the fact that indel‐bearing strands do not exclusively anneal with wild‐type strands[77]. There are several commercially available endonucleases that can be used for the EMC assay–Surveyor™ nuclease[78], T7 endonuclease I[76], CEL‐I endonuclease[79], T4 endonuclease VII[80], and endonuclease V[81]. The endo­nuclease should be carefully selected to suit the application as each enzyme has it is own properties that can impact the sensitivity, efficiency, and specificity of the EMC assay. The method used to separate the products of the cutting reaction can also impact the assay’s performance. For example, up to 3% editing can be detected using Surveyor™ nuclease and WAVE HSD HPLC[78] while up to 0.5% editing can be detected using T7 endonuclease I and gel electrophoresis in polyacryla­mide gel[82].

deletions in the same clone[83]. TIDE and ICE software are designed specifically for CRISPR and do not allow analysis for indels introduced by other genome editors.

Indel Detection by Amplicon Analysis (IDAA) takes advantage of the ability of capillary electrophoresis to resolve small size differences between PCR ampli­cons [87] (Figure 14.5F). The optimized PCR labeling workflow was able to achieve resolution down to a single base‐pair[88]. In IDAA, the region of interest containing the edited site is PCR amplified using three primers‐ two of which are standard gene‐specific primers and the third is a 5′ fluorescently‐labeled universal primer complementary to an overhang on the forward primer. PCR reaction is carried out using touchdown PCR conditions with optimized primers ratio to yield uniformly labeled PCR amplicons with high purity. The labeled amplicons are then resolved using standard capillary electrophoresis and peaks are called using standard capillary electrophoresis software (for detailed protocol please see Ref.[89]). IDAA showed superior performance compared to EMC–IDAA is more sensitive, provides information about indel size, able to detect indels in homozy­gous clones. IDAA showed similar results when compared to NGS with a reported sensitivity of 0.1–1%[89]. Similar to TIDE, IDAA miscalled indels in multiple clones containing both insertions and deletions[83]. In addition, IDAA was not always able to call −1 and +1 nt peaks from the wild‐type peak, but it was possible to do by manual inspections of the peaks[83]. This technique can be a cost‐effective alternative to NGS when the exact sequence of the indels is not required.

engineered nucleases type[11, 90]. Translocations can occur when two or more DSBs are present on the same or two different chromosomes. In this case, one DSB is introduced intentionally by an engineered nuclease acting at a therapeutic site while another DSB is ectopic and introduced due to off‐target activities. Translocations were also detected with only one DSB at the on‐target location when the target region contained substantial sequence homology to another region located elsewhere in the genome[11, 12]. Large chromosomal deletions, inversions, and chromothripsis were also detected at the intended editing sites[11, 12, 14, 91]. Several studies showed that such mutations at the intended editing site may lead to detrimental effects in mitotically active cells[11, 14].

potential structural and numerical chromosome aberrations following genome editing. Newer techniques incorporate fluorescence in situ hybridization (FISH) to identify and evaluate chromosomal rearrangements. Additionally, a soft agar transformation assay using human fibroblasts has been used to assess potential tumorigenicity risk of edited cells[92]. Karyotyping provides a high‐level view of chromosome aberrations, it has a fast turnaround time and is cost‐effective. Though karyotyping is still widely used in industry, it has low sensitivity due to limited resolution and the low number of events that can be screened in one sample.

junction. The 5′‐biotinylated copy is then captured on streptavidin magnetic beads, ligated with bridge adapters followed by two rounds of nested PCR with adaptor and locus‐specific primers that add sample‐specific barcodes and sequences necessary for Illumina sequencing. After the first PCR step, a restric­tion enzyme is used to reduce the background from unrearranged targetalleles. Final libraries are sequenced using Illumina’s MiSeq platform. A bioinformatic pipeline then identifies and maps translocation junctions. This method is designed to detect off‐target editing and unbalanced chromosomal aberrations and meas­ure their relative frequencies. Yin etal. modified LAM‐HTGTS by decreasing number of linear amplification cycles from 80 to 1, introducing ligation with 14‐base pair random UMIs labeling each target copy, deeper sequencing with HiSeq, and improving bioinformatic analysis. This method was named primer‐ extension‐mediated sequencing (PEM‐seq)[95, 96]. The introduction of molecu­lar barcodes allowed to distinguish PCR duplicates from the original templates and together with improved bioinformatics analysis increased the assay sensitiv­ity. PEM‐seq can be applied for measuring short indels. The high amount of gDNA (20–100

All the techniques described above can be used for structural characterization and the discovery of large chromosomal rearrangements. Secondary techniques