Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9–donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9–donor conjugation in addition to delivery influence HDR efficiency.
Get full access to this article
View all access options for this article.
References
1.
MiyaokaY, BermanJR, CooperSB, et al.Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep, 2016; 6:2354–9. DOI: 10.1038/srep23549.
2.
MaliP, YangL, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science, 2013; 339:823–826. DOI: 10.1126/science.1232033.
3.
CongL, RanFA, CoxD, et al.Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339:819–823. DOI: 10.1126/science.1231143.
4.
SchwankG, KooB-K, SasselliV, et al.Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 2013; 13:653–658. DOI: 10.1016/j.stem.2013.11.002.
5.
Van TrungChu, WeberT, WefersB, et al.Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 2015; 33:543–548. DOI: 10.1038/nbt.3198.
6.
LinS, StaahlBT, AllaRK, et al.Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife, 2014; 3:e0476–6. DOI: 10.7554/eLife.04766.
7.
HoltSE, AisnerDL, ShayJW, et al.Lack of cell cycle regulation of telomerase activity in human cells. Proc Natl Acad Sci U S A, 1997; 94:10687–10692.
8.
LeeK, ConboyM, ParkHM, et al.Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng, 2017; 337:1. DOI: 10.1038/s41551-017-0137-2.
9.
YanikM, MüllerB, SongF, et al.In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies. Prog Retin Eye Res, 2017; 56:1–18. DOI: 10.1016/j.preteyeres.2016.09.001.
10.
ZhangJ-P, LiX-L, LiG-H, et al.Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 2017; 18:3–5. DOI: 10.1186/s13059-017-1164-8.
11.
RichardsonCD, RayGJ, DeWittMA, et al.Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol, 2016; 34:339–344. DOI: 10.1038/nbt.3481.
12.
ShibataM, NishimasuH, KoderaN, et al.Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nat Comms, 2017; 8:143–0. DOI: 10.1038/s41467-017-01466-8.
13.
KimS, KimD, ChoSW, et al.Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res, 2014; 24:1012–1019. DOI: 10.1101/gr.171322.113.
14.
KouranovaE, ForbesK, ZhaoG, et al.CRISPRs for optimal targeting: delivery of CRISPR components as DNA, RNA, and protein into cultured cells and single-cell embryos. Hum Gene Ther, 2016; 27:464–475. DOI: 10.1089/hum.2016.009.
15.
LinMI, PaikE, MishraB, et al.CRISPR/Cas9 genome editing to treat sickle cell disease and B-thalassemia: re-creating genetic variants to upregulate fetal hemoglobin appear well-tolerated, effective and durable. Blood, 2017; 130:28–4.
16.
LeeK, MackleyVA, RaoA, et al.Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife, 2017; 6:147–9. DOI: 10.7554/eLife.25312.
17.
KangYK, KwonK, RyuJS, et al.Nonviral genome editing based on a polymer-derivatized CRISPR nanocomplex for targeting bacterial pathogens and antibiotic resistance. Bioconjug Chem, 2017; 28:957–967. DOI: 10.1021/acs.bioconjchem.6b00676.
18.
WenY, PanS, LuoX, et al.A biodegradable low molecular weight polyethylenimine derivative as low toxicity and efficient gene vector. Bioconjug Chem, 2009; 20:322–332. DOI: 10.1021/bc800428y.
19.
BoussifO, Lezoualc'hF, ZantaMA, et al.A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A, 1995; 92:7297–7301.
20.
KhalilIA, KogureK, AkitaH, et al.Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev, 2006; 58:32–45. DOI: 10.1124/pr.58.1.8.
21.
OhY-K, SuhD, KimJM, et al.Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther, 2002; 9:1627–1632. DOI: 10.1038/sj.gt.3301735.
22.
PollardH, RemyJS, LoussouarnG, et al.Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J Biol Chem, 1998; 273:7507–7511. DOI: 10.1074/jbc.273.13.7507.
23.
DrakeDM, PackDW. Biochemical investigation of active intracellular transport of polymeric gene-delivery vectors. J Pharm Sci, 2008; 97:1399–1413. DOI: 10.1002/jps.21106.
24.
SuhJ, WirtzD, HanesJ. Efficient active transport of gene nanocarriers to the cell nucleus. Proc Natl Acad Sci U S A, 2003; 100:3878–3882. DOI: 10.1073/pnas.0636277100.
25.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337:816–821. DOI: 10.1126/science.1225829.
26.
Carlson-StevermerJ, AbdeenAA, KohlenbergL, et al.Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat Comms, 2017; 8:171–1. DOI:10.1038/s41467-017-01875-9.
27.
JinekM, JiangF, TaylorDW, et al.Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014; 343:1247997–1247997. DOI: 10.1126/science.1247997.
28.
NishimasuH, RanFA, HsuPD, et al.Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 2014; 156:935–949. DOI: 10.1016/j.cell.2014.02.001.
29.
YuX, LiangX, XieH, et al.Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnol Lett, 2016; 38:919–929. DOI: 10.1007/s10529-016-2064-9.
30.
AkitaH, ItoR, KhalilIA, et al.Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy. Mol Ther, 2004; 9:443–451. DOI:10.1016/j.ymthe.2004.01.005.
31.
CardarelliF, DigiacomoL, MarchiniC, et al.The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery. Sci Rep, 2016; 6:2587–9. DOI: 10.1038/srep25879.
32.
CertoMT, RyuBY, AnnisJE, et al.Tracking genome engineering outcome at individual DNA breakpoints. Nat Meth, 2011; 8:671–676. DOI: 10.1038/nmeth.1648.
33.
RobertF, BarbeauM, ÉthierS, et al.Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med, 2015; 7:9–3. DOI: 10.1186/s13073-015-0215-6.
34.
OgrisM, SteinleinP, KursaM, et al.The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther, 1998; 5:1425–1433. DOI: 10.1038/sj.gt.3300745.
35.
AdachiO, NakanoA, SatoO, et al.Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis. Gene Ther, 2002; 9:577–583. DOI: 10.1038/sj.gt.3301691.
36.
JiangC, DavalosRV, BischofJC. A review of basic to clinical studies of irreversible electroporation therapy. IEEE Trans Biomed Eng, 2015; 62:4–20. doi:10.1109/TBME.2014.2367543
37.
MaM, ZhuangF, HuX, et al.Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res, 2017; 27:578–581. DOI: 10.1038/cr.2017.29.
38.
GuB, PosfaiE, RossantJ. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol, 2018; 36:632–637. DOI: 10.1038/nbt.4166.
39.
AirdEJ, LovendahlKN, MartinAS, et al.Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol, 2018; 1:5–4. DOI: 10.1038/s42003-018-0054-2.
40.
SavicN, RingnaldaFC, LindsayH, et al.Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. eLife, 2018; 7:16–3. DOI: 10.7554/eLife.33761.
41.
LimKH, HuangH, PralleA, et al.Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol Bioeng, 2013; 110:57–67. DOI: 10.1002/bit.24605.
42.
GuschinDY, WaiteAJ, KatibahGE, et al.A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 2010; 649:247–256. DOI: 10.1007/978-1-60761-753-2_15.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
