CRISPR-Cas9 Genome Editing

The assignment discusses the creation of parental cell lines and mono-allelic knockout cells derived from C6/36 cells. The Odd3 clones show mixed populations with mono-allelic mutations, while the 4G5 clones indicate the presence of deletions. Gel Electrophoresis results confirm the presence of insertions and deletions in some samples. The TIDE method was used to determine and predict the mutation size, which was confirmed by sequencing and CRISP-ID detection. The analysis reveals that some cells retained the parent allelic mutation, while others showed non-functional proteins due to mutations.

C6/36 is the parental cell line, which was derived from larvae of the Asian tiger mosquito, Aedes albopictus. The
clones Odd3 and 4G5, 2nd generation derived from C6/36 cells. Mono-allelic knockout cells have been created, as
shown in figure (Figure 6). Then, a second CRISPR knockout has been performed on these mono-allelic knockout cells
to 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 analysis
shows that the retaining of the parent (Odd3) allelic mutation that characterised by the presence of 5 nucleotide
insertion (Figure8). On the other hand, Odd3-1C3 is sub-population of Odd3 with a monoallelic mutation that has four
nucleotides deletions (Figure8).
4G5 clones indicate the presence of mono-allelic mutations due to presence of deletions in two sites, 24 and 22
nucleotides’ deletions (Figure8). Interestingly, 24 nucleotides’ deletion was in-frame deletions. Therefore, the exon-1
and protein function is retained. 4G5-4B3, cells generated from the 4G5 cells, was screened and analysed. The
screening 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 parent
cell 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 the
deletions. Additionally, 4G5 cells and 4G5-4B3 gel showed the presence of deletions. Thus, identifying and predicting
the mutation and its size is necessary.
Tracking of Indels by Decomposition (TIDE) method was used to determine and predict the mutation. TIDE
interpretation confirmed what is seen in the gel electrophoresis. TIDE analysis (Figure 8) shows the presence of five
nucleotide insertion and four nucleotide deletions in Odd3; Five nucleotide insertions in Odd3-4E4; and four
nucleotide deletions in Odd3-1C3 compared to the parent sequence (C6/36). Also, 4G5 and 4G5-4B3 analysis predicted
the 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 Odd3-1C3
shows 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 mixed
sequences. The mixed sequences were compared to the reference sequence, and two allelic sequences were provided
distinctly for Odd3, Odd3-4E4, and Odd3-1C3, while three allelic sequences for 4G5 (Figure10).

Then Clustal Omega Alignments is used to alignments of sequences from CRISPR-ID with the parent sequence to
determine the Indels position. Alignments of Odd3 and Odd3-4E4 show the presence of 5 bp insertion (Figure 11 and
12), while Alignments of Odd3-1C3 shows the presence of 4 bp deletions (Figure13). The alignment of 4G5 indicates
the occurrence of 22 bp deletion and 24 bp (Figure14).
The sequences of Nucleotides were translated to look for potential changes in exon-1 (yellow coloured). The correct
reading frames were determined for Odd3, Odd3-4E4, Odd3-1C3, and 4G5 (Figure 15), which is started with initiating
codon atg (Methionine) and ends with a stop codon.
Exon-1 is linked to the rest of the protein sequence to check the change in the entire Phb-2 protein. To do this, ExPASy
Translate is used to translate the Exon-1 of the parent, allelic-1, and allelic-2 with the rest of the nucleotide sequence.
Then, the changes were checked in the entire sequence through aligned of the all three proteins that have different
exon-1 to the parent sequence.
The odd3 alignment shows a change in the entire Phb-2 sequence only of allelic-2 resulting in a nonfunctional protein
(figure 20). In contrast, allelic-1 shows no variations in the whole Phb-2 gene (mono-allelic mutation). Odd3-4E4 is
same as Odd3 retained the parent mono-allelic mutation (figure 21). Also, Clustal alignment of Odd3-1C3 proteins
shows a nonfunctional protein in only allelic-2 sequence as Odd3 with mono-allelic mutation (figure 22). In addition,
4G5 alignments show a change in the entire Phb-2 sequence only of allelic-3 that leading to a nonfunctional protein
(figure 23).
The analysis of two distinct cell lines derived from the parent C6/36 cell concluded a mono-allelic knockdown in cell
lines. Odd3 3rd generation cells retained the parent allelic mutation. The analysis of 4G5 cell line revealed obtaining an
in-frame shifting as a result of a deletion of 24 nucleotides that does not cause loss of prohibition-2 function.