Tracking of Indels by Decomposition (TIDE) Method
Added on - 17 Oct 2019
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C6/36 is the parental cell line, which was derived from larvae of the Asian tiger mosquito, Aedes albopictus. Theclones Odd3 and 4G5, 2nd generation derived from C6/36 cells. Mono-allelic knockout cells have been created, asshown in figure (Figure 6). Then, a second CRISPR knockout has been performed on these mono-allelic knockout cellsto produce a bi-allelic knockout. The cloned cells, 3rd generation cells, are screened for the bi-allelic knockout.Odd3 shows mixed population with mono-allelic mutations (Figure8). The mixed populations are a major population(41.3%) with insertion and a minor population (2.6 %) with deletion Thus, Odd3-4E4 is re-edited. The data analysisshows that the retaining of the parent (Odd3) allelic mutation that characterised by the presence of 5 nucleotideinsertion (Figure8). On the other hand, Odd3-1C3 is sub-population of Odd3 with a monoallelic mutation that has fournucleotides deletions (Figure8).4G5 clones indicate the presence of mono-allelic mutations due to presence of deletions in two sites, 24 and 22nucleotides’ deletions (Figure8). Interestingly, 24 nucleotides’ deletion was in-frame deletions. Therefore, the exon-1and protein function is retained. 4G5-4B3, cells generated from the 4G5 cells, was screened and analysed. Thescreening indicated keeping the same mutations as 4G5 cells.Gel Electrophoresis results (Figure 7) of the Odd3 band shows a slight variation in size when compared to the parentcell C6/36. The Odd3 band width is larger than parent suggesting the presence of insertions as well as deletions. Also,Odd3-4E4 bands indicated the presence of insertions. Moreover, Odd3-1C3 bands indicated the presence of thedeletions. Additionally, 4G5 cells and 4G5-4B3 gel showed the presence of deletions. Thus, identifying and predictingthe mutation and its size is necessary.Tracking of Indels by Decomposition (TIDE) method was used to determine and predict the mutation. TIDEinterpretation confirmed what is seen in the gel electrophoresis. TIDE analysis (Figure 8) shows the presence of fivenucleotide insertion and four nucleotide deletions in Odd3; Five nucleotide insertions in Odd3-4E4; and fournucleotide deletions in Odd3-1C3 compared to the parent sequence (C6/36). Also, 4G5 and 4G5-4B3 analysis predictedthe presence of 22 and 24 bp deletions with 23.9% and 53 % mutation efficacy respectively.Cells were sequenced to identify indels. Therefore, Sequences chromatogram of Odd3, Odd3-4E4, and Odd3-1C3shows mixed sequences (Figure 9). Additionally, Indels identification is difficult to describe with overlapped peaks.Thus, CRISP-ID detected the correct Indels size and targeted region of CRISPR/Cas9 allowing distinguishing mixedsequences. The mixed sequences were compared to the reference sequence, and two allelic sequences were provideddistinctly for Odd3, Odd3-4E4, and Odd3-1C3, while three allelic sequences for 4G5 (Figure10).