Analysis of CRISPR/ Cas9 Mutations

The parental cell line C6/36 was generated from Asian tiger mosquito larvae, and mono-allelic knockout cells were developed by CRISPR/Cas9 gene editing. The study aimed to investigate the effects of mono-allelic mutations on the PHB-2 protein sequence. Three cell lines, Odd3, Odd3-4E4, and 4G5, were analyzed for their nucleotide sequences, protein modifications, and functional changes. The results showed that Odd3 and its sub-populations exhibited insertions and deletions, while 4G5 demonstrated in-frame deletions. The study concluded that the mono-allelic mutations did not affect the functioning of the restriction-2 enzyme in 4G5 cells.

The parental cell line C6/36 was generated from the Aedes albopictus, Asian tiger mosquito’s larvae. The
2nd generation clones Odd3 and 4G5 were obtained from C6/36 cells. As shown in figure (Figure 6)
mono-allelic knockout cells have been developed; after that, to originate a bi-allelic knockout a second
CRISPR knockout obsolete accomplished upon these mono-allelic knockout cells. Also, the 3rd
generation cloned cells are assessed to generate the bi-allelic knockout.
Odd3 indicates varied populace accompanying mono-allelic mutations (Figure8). The varied populace
consists of majorly with insertion (41.3%) and inconsiderably with deletion (2.6 %). Therefore, the data
analysis demonstrates that the re-editing takes place for Odd3-4E4 as the parent (Odd3) conserved the
allelic mutation which is represented by the insertion of 5 nucleotides (Figure8). Then again, Odd3 has
the sub-population naming Odd3-1C3 characterized by mono-allelic mutation with deletion of four
nucleotides (Figure8).
4G5 clones demonstrate the existence of mono-allelic mutations because of the occupancy of 24 and 22
nucleotides’ deletions in two respective sites (Figure8); concernedly, the deletion of 24 nucleotides’ was
designated as in-frame deletions. Subsequently, the exon-1 along with the functioning of protein is
retained. 4G5-4B3 cells, produced from the 4G5 cells were examined and screened to demonstrate the
retention of similar mutations likewise 4G5 cells.
Odd3 band Gel Electrophoresis (Figure 7) result demonstrates a minor difference in size in comparison
with the parent cell C6/36. The larger bandwidth of Odd3 than its parent recommending the existence
of both insertions and deletions. Additionally, Odd3-4E4 bands showed the existence of insertions.
Besides, Odd3-1C3 bands demonstrated the occurrence of deletions. Moreover, 4G5 cells and 4G5-4B3
cells both demonstrated the existence of deletions. Therefore, distinguishing plus anticipating the
presence of a mutation and estimate its size is fundamental.
Tracking of Indels by Decomposition (TIDE) technique usually utilized to decide and anticipate the
mutation also help to affirm the findings of gel electrophoresis. TIDE analysis (Figure 8) demonstrates
the presence of insertion of five nucleotides and deletion of four nucleotides in Odd3; additionally,
insertion of five nucleotides in Odd3-4E4; also deletion of four nucleotides in Odd3-1C3 contrasted with
the parent C6/36 sequence. Likewise, analysis of 4G5 and 4G5-4B3 anticipated the existence of deletions
of 22 and 24 bp accompanied by 23.9% and 53 % mutation viability separately.
To recognize indels the cells needed to be sequenced. Thus, sequencing chromatogram of Odd3, Odd3-
4E4, and Odd3-1C3 display diverse sequences (Figure 9). Also, Indels recognizable proof somewhat hard

to portray alongside overlapped peaks. Consequently, CRISP-ID identified the exact size of Indels and the
target region of CRISPR/Cas9 and also permitted to recognize diverse sequences. The diverse sequences
were contrasted with the reference sequence; whereas, for Odd3, Odd3-4E4, and Odd3-1C3 two allelic
sequences and for 4G5 three allelic sequences were dispensed markedly (Figure10).
After that Clustal Omega alignment is utilized for the positioning of sequences derived from CRISPR-ID
including its parent sequence to determine the position of Indels. Odd3 and Odd3-4E4 both alignments
demonstrate the existence of the deletion of 5 bp (Figure13), while Odd3-1C3 alignments demonstrate
the existence of deletion of 4 bp (Figure13). The 4G5 alignment shows the presence of deletions of 22
bp and 24 bp respectively (Figure14).
The nucleotide sequences were translated to figure out the possible modifications in exon-1 (yellow
colored). The correct reading frames which are known, to begin with, initiation codon ATG and finished
up with a stop codon were resolved for Odd3, Odd3-4E4, Odd3-1C3, and 4G5 (Figure 15).
Exon-1 is connected to the remaining protein sequence for the complete analysis of the PHB-2 protein
modification and to perform this analysis ExPASy Translate is utilized for the translation of Exon-1 of the
parent, allelic-1, and allelic-2 accompanying the remaining nucleotide sequence. At that point, the
progressions were investigated for the whole genome sequence by the alignment of the each of the
three proteins which possess diverse exon-1 from their parent sequence.
The Odd3 alignment demonstrates a modification in the whole PHB-2 sequence except that allelic-2
brings about a non-functional protein (figure 20). Conversely, allelic-1 demonstrates no any alteration in
the entire PHB-2 gene (mono-allelic mutation). Odd3-4E4 has the similarity like Odd3 as both conserved
the parent mono-allelic mutation (figure 21). Additionally, Odd3-1C3 Clustal alignment demonstrates
the presence of nonfunctional protein exclusively in allelic-2 sequence because Odd3 consist of mono-
allelic mutation (figure 22). Likewise, 4G5 alignments demonstrate a modification within whole PHB-2
sequence except in case of the allelic-3 which prompting in the development of a nonfunctional protein
(figure 23).
The evaluation of two particular cell lines which was obtained from the parent cell (C6/36) resulting in
the mono-allelic knockdown among cell lines. Third era cells of Odd3 conserved the allelic mutation
from its parent. The evaluation of 4G5 cell line exhibited the acquiring of in-frame shifting mutation due
to the outcome of the deletion of 24 nucleotides that does not promote deprivation of functioning of
the restriction-2 enzyme.