Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 16:7:53-60.
doi: 10.1016/j.omtn.2017.02.006. Epub 2017 Feb 28.

Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks

Fei Song  1 , Knut Stieger  2
Affiliations

Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks

Fei Song et al. Mol Ther Nucleic Acids. .

Abstract

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated proteins) technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. In this study, we analyzed the impact of different repair templates on the frequency of homology-directed repair (HDR) and non-homologous end joining (NHEJ). We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to quantify HDR and NHEJ events following transfection with Cas9, eight different guide RNAs, and a 1,000 bp donor template generated either as circular plasmid, as linearized plasmid with long 3' or 5' backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5' homologous region (RS37), or with a shorter 3' homologous region (RS73). Guide RNAs _targeting the transcriptionally active strand (T5, T7) showed significantly higher NHEJ frequencies compared with guide RNAs _targeting the transcriptionally inactive strand. HDR activity was highest when using the linearized plasmid with the short 5' backbone overhang and the RS37 design. The results demonstrate the importance of the design of the guide RNA and template DNA on the frequency of DNA repair events and, ultimately, on the outcome of treatment approaches using HDR.

Keywords: CRISPR-Cas9; DNA donor template; HDR; NHEJ.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Design of CRISPR-Cas9 Guide RNAs _targeting TLR3 Sequence _targets (T3–T8) around the stop codon and I-SceI site (yellow line) have been designed for the specific cleavage within the GFP sequence in the TLR3 system. Depending on the addition of the donor templates, DSBs can be repaired through either NHEJ (BFP) or HDR (GFP). T, _target.
Figure 2
Figure 2
Activity of the CRISPR-Cas9 _targets in the TLR3 System (A) Guide RNAs are cloned into the px459 vector under the hU6 promoter. (B) px459 Cas9 _targets were expressed in the HEK-TLR3 cell line. Seventy-two hours after transfection, flow cytometric analysis displayed about 27% of BFP+ (NHEJ repair) cells by px-459-T7. The graphs represent triplicate data from three independent experiments. Statistical analysis: ANOVA, ***p < 0.001. (C) Analysis of sequence modifications after T3 cleavage. Sequence changes were analyzed using Sanger sequencing via TOPO Cloning. Sequence in red: PAM site; sequence in blue: guide RNA _target sequence; sequence in yellow: changes to the wild-type sequence. T0, px459 without guide RNA; T3, _target 3.
Figure 3
Figure 3
Generation of Different Donor Templates and px459-mRFP _target (A) One kilobase of double-stranded DNA (dsDNA) donor templates was generated with varying homology sequence overlaps on the 5′ and 3′ side of the mutation site. (B) Donor templates were generated as plasmid, linearized plasmid with 5′ or 3′ backbone overhang, or PCR product. (C) mRFP gene sequence was cloned into the px459 expression vector instead of the puromycin resistance gene.
Figure 4
Figure 4
FACS Analysis of HDR and NHEJ Events Using the Circular Plasmid RS55 (A) The TLR3 plasmid was cotransfected with nucleases and donor template for the quantification of both HDR and NHEJ events. Artificial NHEJ (TLR3-delTA) and HDR (TLR3-correctGFP) control for the TLR systems were generated through deletion of 2 bp nucleotides (thymin and adenine) and correction of mutant sequence, respectively. (B) FACS data of the respective samples and controls. Statistical analysis: paired t test, NHEJ values compared with T0 plasmid transfection, HDR values compared with RS55 plasmid alone. *p < 0.001; **p < 0.0001; ***p < 0.00005.
Figure 5
Figure 5
Optimizing HDR Events Using Different Donor Templates Cas9-mRFP _targets and donor templates were coexpressed in the HEK-TLR3 cell line. (A–F) Seventy-two hours after transfection, flow cytometric analysis of mRFP+ gated cells displayed GFP+ (A, C, and E, HDR repair) and BFP+ (B, D, and F, NHEJ repair) cells. The graphs represent triplicate data from three independent experiments. lin., linearized; Pla., plasmid; RS, repair substrate; T0, px459-mRFP without guide RNA. Statistical analysis: ANOVA, *p < 0.05; **p < 0.005; ***p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Barrangou R., Fremaux C., Deveau H., Richards M., Boyaval P., Moineau S., Romero D.A., Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315:1709–1712. - PubMed
    1. Barrangou R., van Pijkeren J.P. Exploiting CRISPR-Cas immune systems for genome editing in bacteria. Curr. Opin. Biotechnol. 2016;37:61–68. - PubMed
    1. Chang N., Sun C., Gao L., Zhu D., Xu X., Zhu X., Xiong J.W., Xi J.J. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 2013;23:465–472. - PMC - PubMed
    1. DiCarlo J.E., Chavez A., Dietz S.L., Esvelt K.M., Church G.M. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat. Biotechnol. 2015;33:1250–1255. - PMC - PubMed
    1. Friedland A.E., Tzur Y.B., Esvelt K.M., Colaiácovo M.P., Church G.M., Calarco J.A. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods. 2013;10:741–743. - PMC - PubMed
  NODES
twitter 2