CRISPR/Cas9-mediated site specific integration of LP onFer1L4
Genomic hotspot is a locus that allows for stable and strong transgene
expression (Nathaniel K. Hamaker & Kelvin
H. Lee, 2018). Fer1L4 has been identified as genomic hotspot in
CHO cells (Zhang et al., 2015). Hence, we
selected this region as a targeted integration site. LP was designed
where Lox5171/LoxP sites flanked mCherry followed by CMV promotor-driven
thymidine kinase (TK ) gene (Fig. 1A). It also contained the 5′ HA
(0.2 kb region of Fer1L4 exon 1 present before the transcription
start site) and 3′ HA (0.2kb region of Fer1L4 exon 1 present
after the transcription start site) (Fig. 1A). For RMCE-mediated gene
swapping, TV was designed where Lox5171/LoxP sites flanked GFP, followed
by an SV40 promoter-driven neomycin resistance gene (Fig. 1B).
We then performed CRISPR/Cas9-mediated homologous recombination to
integrate LP into the Fer1L4 exon 1 region (Fig. 2A). Fourteen
days after drug selection, single cell clones were isolated using
limiting dilution in 96-well plates, and 12 clones expressing mCherry
were seleced (Fig. 2B). We then examined how many copies ofFer1L4 were targeted by LP. A Fer1L4 forward (Fwd) primer
was designed to bind to the sequence before the 5′ HA, and aFer1L4 reverse (Rev) primer was designed to bind to the sequence
after the 3′ HA (Fig. 2C). When Fer1L4 is not targeted by LP, a
200 bp PCR product will be amplified (Fig. 2C). However, whenFer1L4 is targeted by LP, a 3,300 bp PCR product will be
amplified (Fig. 2C). Therefore, to amplify only the Fer1L4 not
targeted by LP, qPCR was performed with a 5 s extension time. Genomic
DNA from CHO cells was used as a negative control (Fig. 2D). Eight of
the 12 clones showed half the value compared to the negative control,
indicating that only one of the two copies of Fer1L4 was targeted
by LP (Fig. 2D; arrowhead).
We then examined the nucleotide sequence of the recombinant region. To
amply the Fer1L4 region targeted by LP, nested PCR was performed
on genomic DNA with 3 sets of primers (Fig. 3A). The
1st Fwd primer was designed to bind to the sequence
before the 5′ HA, and the 1st Rev primer was designed
to bind to the mCherry sequence (Fig. 3A). The 1st PCR
amplification using the 1st primer set generated a
template for a 2nd PCR (Fig. 3A and B). Subsequently,
the 2nd PCR amplification with the
2nd primer set generated a template for a
3rd PCR (Fig. 3A and B). Finally, the
3rd PCR amplification using the 3rdset of primers generated a 348 bp amplicon in clone #11 (Fig. 3B; red
arrow). However, other clones (clones #4, #6, #8 and #9) were found
to have undergone LP integration in Fer1L4, but did not yield the
348 bp amplicon (Fig. 3B). These results indicated that the quality of
genomic DNA extracted from those clones was not sufficient to generate
PCR amplicons. However, further verification is needed to clarify this
possibility. We then performed DNA sequencing on the amplified PCR
products from clone #11, and found that the sequence of the PCR product
was identical to that of the Fer1L4 region targeted by LP. This
result suggests site-specific LP targeting in Fer1L4 (Fig. 3C).