Homology-mediated end joining-based targeted integration using CRISPR/Cas9

Abstract

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

CRISPR‐Cas system: Toward a more efficient technology for genome editing and beyond

“…The researchers showcased homology‐mediated gene integration utility by designing the new homology‐mediated end joining (HMEJ) donor sequence that requires sgRNA target sites as well as ~800 bp homology arms. The HMEJ‐based strategy yielded a high penetrance gene transmission than existing strategies in multiple systems . More importantly, this approach achieved gene‐edited monkeys by coinjection of different volumes and concentrations of donor plasmid together with Cas9 mRNA .…”

“…The efficiency of targeted transgenes knock-in illustrated by recent reports, further highlighting the broad promise of CRISPR-Cas9-mediated PITCh (CRIS-PITCh) systems via microhomology-mediated end joining (MMEJ). 15,19,24,25 This method utilities original CRIS-PITCh vector (v1) and upgraded PITCh version (CRIS-PITCh (v2)) for external DNA knock-in ( Figure 1A,B). For a practical application, CRIS-PITCh-v2 system was applied to transfer anti-prion scFv-Fc antibody into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cell lines.…”

Making ends meet: targeted integration of DNA fragments by genome editing

“…Interestingly, the efficiency of insertion of 714 bp in human cells was the same regardless if the homology arms were 33 bases or 518 bases [Paix et al 2017a]. This is in contrast to the HMEJ experiments described above [Yao et al 2017] where long homology arms perform better than short homology arms. A detailed protocol on performing edits of either DNA insertions or deletions using short homology arms has recently been published and contains useful tips [Paix et al 2017b].…”

CRISPR editing in biological and biomedical investigation

“…Inspired by this, we generated site‐specific Uncoupling protein 1 ( UCP1 ) knock‐in pigs efficiently and reconstituted UCP1 expression in porcine white adipose tissue via such HITI strategy (unpublished data). Recently, microhomology‐mediated end joining (MMEJ)‐ and homology‐mediated end joining (HMEJ)‐based strategies are reported to have the capable of integrating long exogenous DNA fragments into the genome at relatively high frequencies (Figure a) (Nakade et al, ; Yao et al, ), which suggested that these HDR dependent and independent strategies are most sensible to introduce site‐specific transgene integration without the cell cycle dependence and is free from the exogenous molecule cytotoxicity.…”