A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

Abstract

Abbreviations: bGHpA, bovine growth hormone polyadenylation signal; Cas9, CRISPRassociated protein 9; CHO, Chinese hamster ovary; CLD, cell line development; CRISPR, clustered regularly interspaced short palindromic repeats; DSB, double-strand break; eGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; gRNA, guide RNA; hCMV, human cytomegalovirus major immediate-early promoter; HDR, homologydirected repair; HMEJ, homology-mediated end joining; INDEL, insertion or deletion; KO, knockout; LP, landing pad; MMEJ, microhomology-mediated end joining; NHEJ, nonhomologous end joining; PAM, protospacer adjacent motif; PEST, sequence rich in proline,

High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

“…The SSIGNAL reporter system was previously established to measure gene disruption and SSI using exogenous DNA donors (Hamaker & Lee, 2020). Here, we wanted to develop a new reporter that preserved the characteristics of the two‐color SSIGNAL reporter system, while also expanding upon its capabilities by introducing a third fluorescent marker to indicate RI.…”

“…We previously integrated the chromosomal component of the SSIGNAL reporter system at the CHO Rosa26 locus, which was configured with orthogonal Bxb1 integrase recognition sites (i.e., attP/attP′ ) flanking the dGFP coding sequence (Hamaker & Lee, 2020). This landing pad functionality was meant to facilitate RMCE according to previously developed methods (Inniss et al, 2017).…”

“…The split sfOrange coding sequence was purchased as a gBlock (IDT) and cloned into a mammalian expression plasmid to make pLP1‐cbsfOinsb. The pLP1 expression plasmid, which has a human cytomegalovirus (hCMV) major immediate‐early promoter and bovine growth hormone polyadenylation signal (bGHpA), was described previously (Hamaker & Lee, 2020).…”

“…Rapidly dividing cell types exhibit SSI efficiencies that rarely exceed 10%, and 1% or less is typical when standard homology‐directed repair (HDR) approaches are utilized in CHO cells (Bosshard et al, 2019; Hamaker & Lee, 2020; Inniss et al, 2017; Lee et al, 2016). Notably, recent work has demonstrated significant improvement by using modified HDR donor formats, namely double‐cut donors (DCDs), where CRISPR‐mediated donor plasmid linearization and formation of the chromosomal DSB occur synchronously (Shin & Lee, 2020b; Yao et al, 2017; J.‐P.…”

CRISPR‐interceded CHO cell line development approaches

“…Alternatively, by optimizing the CRISPR‐Cas9 procedure (e.g., evaluating different sgRNAs and achieving high transfection efficiency), Pourtabatabaei et al (2021) achieved a high integration rate to target a 1.8 kb gene cassette in the CHO‐K1 cell line. Fluorescent enrichment is also another trick that has been used to enhance KI efficiency (Hamaker & Lee, 2020; J. S. Lee et al, 2016). It is now well recognized that the molecular mechanism behind plasmid‐based integration is homology‐directed repair (HDR) that executes the gene integration precisely without any erroneous insertions and deletions (indels) at locus‐transgene boundaries (Ran et al, 2013).…”