HMEJ-mediated efficient site-specific gene integration in chicken cells

Abstract

Background: The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results: Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system. Conclusions: Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

Novel Therapeutic Approaches for the Treatment of Retinal Degenerative Diseases: Focus on CRISPR/Cas-Based Gene Editing

“…In another study, the HMEJ strategy was compared side-by-side with the HITIand HDR-based gene editing methods in chicken DF-1 and primordial germ cells. The results from this work showed that HMEJ was the most efficient in mediating precise gene knock-in (Xie et al, 2019). A recent publication introduced another HMEJbased approach (named GeneWeld) in which shorter homology segments of 24-48 base pairs were sufficient to drive precise reporter gene knock-in in zebrafish and mammalian cells using easily engineered donor DNA constructs in which the reporter transgene was flanked by universal gRNA target sequences (Wierson et al, 2020).…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…The MMEJ-based strategy was subsequently devised to an HMEJ approach by enabling more efficient targeted transgene integration using longer and more stable homology arms 27 , 37 – 39 . In this case, CRISPR/Cas9 is designed to cleave both the targeted genomic locus and transgene donor vector that contains long homology arms (600–900 bp each homology arm) ( Figure 3E ).…”

“…The repair template can be in the form of circular double-stranded plasmid DNA 24 – 27 , single-stranded donor oligonucleotide (ssODN) 28 – 30 , linear double-stranded polymerase chain reaction (PCR) fragments 31 , 32 , or the homologous sequences of the intact sister chromatid 33 . Depending on the forms of repair template and CRISPR system used, homology-mediated gene insertion and replacement are carried out via specific DNA repair pathways such as homology-directed repair (HDR) 24 , 26 , 33 , synthesis-dependent strand annealing (SDSA) 28 , 34 , microhomology-mediated end joining (MMEJ) 35 , 36 , and homology-mediated end joining (HMEJ) 27 , 37 – 39 pathways. In homology-independent approaches, transgene knock-in and gene correction are achieved with CRISPR/Cas9 in the absence of DNA donor repair templates.…”

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing

“…In the presence of a donor template harboring microhomology arms (5-40 bp) flanking the genomic target locus, MMEJ could be used to mediate precise insertion of exogenous DNA (Figure 1 C) 48 - 51 . In addition, homology-mediated end joining (HMEJ)-based strategy with long homology arms (~800 bp) was reported to achieve precise gene integration with greater knock-in efficiency than MMEJ-based strategy (Figure 1 C) 52 - 54 .…”