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Latest Discovery   |   Jan 21, 2020

De-arming CRISPR/Cas9 to increase knock-in efficiency

De-Arming CRISPR/Cas9 to increase knock-in efficiency

The ability to knock in (KI) DNA sequences via homologous recombination (HR) to generate knockout (KO) mice was first demonstrated 30 years ago by Mario R. Capecchi, Martin J. Evans and Oliver Smithies (1,2). In recent years, CRISPR/Cas9 has revolutionized the field of genome editing in mammalian cells including generation of KO and KI transgenics (3, 4).  

Despite these recent advances, the efficiency of targeted KI of genes is still low especially in primary cells such as human stem cells (5). Improvement of site-specific KI would clearly benefit a number of functional studies such as reporter/allele-specific gene expression, disease modelling and elucidation of chromatin structure. In a recent paper, Yu et al report on a new method that allows enhanced levels of gene KI by utilizing 5’-modified donor DNA with short homology arms (6). Using linear double-stranded DNA (dsDNA) sequence as donor templates, a number of 5’-end and 3’-end modifications were tested for inserting a GFP fragment into the GAPDH locus in a human colon cancer cell line (HCT116). 13 modifications were tested including phosphorothioate linkages, amine groups and N-hydroxysuccinimide (NHS) esters, all introduced by PCR.

Several 5’ modifications were shown to enhance KI of the reporter gene resulting in 2.3- to 5.1-fold increases in KI efficiencies with a 5’ C6-PEG10 modification being the most effective. CRISPR/Cas9 often requires long (~500 bp) homology arms (HAs) for correct targeting to a specific locus. Here, KI efficiency was shown to be greater with 5’ C6-PEG10 dsDNA containing 50 bp HAs compared to unmodified dsDNA donor sequence with 500 bp HAs (no enhancement was observed using 5’ modified dsDNA with 500 bp HAs) concomitant with lower levels of background EGFP expression resulting from random integration. 5’ C6-PEG10 modified dsDNA with short HAs was further evaluated versus methods in previous studies using similar systems, revealing enhanced KI into the GAPDH locus in HEK293, HCT116, human iPS cells (WTC G3) and human embryonic stem cells (ESCs). KI of EGFP into LMNA encoding lamin A/C in HEK293 cells demonstrated a 65% KI rate using 5’ C6-PEG10 modified donor DNA compared to 15% with unmodified DNA. For generation of transgenic mice and rats, safe harbour genomic sites such as Rosa26, AAVS1 and CCR5 are routinely targeted. 5’ C6-PEG10 modification improved KI rate at both AAVS1 and CCR5 albeit only moderately. Interestingly, adding 5’ C6-PEG10 to 2.5 kb dsDNA resulted in much higher KI levels at the AAVS1 locus. Lastly, multiplexing potential using the 5’ C6-PEG10 modification was examined by analysing dual KI into GAPDH and LMNA loci. Consistently, 5’ C6-PEG10 modified donors promoted a higher rate of EGFP/mCherry expression demonstrating enhanced KI.

In summary, while the mechanism responsible remains poorly understood, 5’ addition of C6-PEG10 to dsDNA results in 5-fold increases in KI efficiency as well as lower indel rates in KI junctions. Although this modification was not examined in the context of transgenic animal generation where larger sequences are required, this is the highest reported KI rate to date and will likely improve many functional studies.


VectorBuilder, through our unique design platform and new manufacturing facility, offers solutions to all your custom CRISPR needs including gRNA vectors, gRNA sensors, CRISPRi and CRISPRa as well as donor vectors. All vectors are available as ready-to-use virus particles including AAV, adenovirus lentivirus, MSCV and MMLV, for both in vitro and in vivo applications. VectorBuilder also offers BAC recombineering, custom stable cell lines and CRISPR/shRNA libraries, all at low cost and with fast turnaround.


  1. Capecchi, M.R. The new mouse genetics: altering the genome by gene targeting. Trends in genetics. 1989;5(3):70-6.
  2. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51(3):503-12.
  3. Banan M. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. Journal of Biotechnology. 2019;308:1-9.
  4. Doudna, J. A. et al. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
  5. Merkle, F. T. et al. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep. 2015;11(6):875-883.
  6. Yu, Y. et al. An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms. Nat Chem Biol. 2019; doi:10.1038/s41589-019-0432-1.
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