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Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

View Article: PubMed Central - PubMed

ABSTRACT

The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

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Correlation between CRISPR/Cas9 cleavage and gene editing activity.Synchronized and released HCT116-19 cells were electroporated with 0.0–10.0 μg of pX330 and with (+ODN) or without (−ODN) 1.35 μg of 72-mer. CRISPR/Cas9 cleavage activity measured by Surveyor endonuclease assay (orange and yellow) as well as gene editing (dark green and light green) activity measured by FACS are shown. Standard error is represented by the bars on the each data point.
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f3: Correlation between CRISPR/Cas9 cleavage and gene editing activity.Synchronized and released HCT116-19 cells were electroporated with 0.0–10.0 μg of pX330 and with (+ODN) or without (−ODN) 1.35 μg of 72-mer. CRISPR/Cas9 cleavage activity measured by Surveyor endonuclease assay (orange and yellow) as well as gene editing (dark green and light green) activity measured by FACS are shown. Standard error is represented by the bars on the each data point.

Mentions: Next, DNA cleavage activity, generated through the action of the CRISPR/Cas9 system, was determined by the Surveyor endonuclease at dosages that support significant levels of gene editing. The data are presented in Fig. 3 and supplementary Figure S1. The Surveyor assay successfully detected CRISPR/Cas9 activity in the absence of the ssODN; a predictable rise in activity was observed as a function of the amount of expression vector present in the reaction. When cleavage activity is measured in complete reaction mixtures, i. e. in the presence of the oligonucleotide, Surveyor endonuclease activity is increased (orange line vs yellow line), particularly at the two doses where gene editing activity is near maximal. While this may appear to be counterintuitive, the Surveyor endonuclease assay has a preference for DNA duplexes with small but definitive indels. DNA duplexes bear small insertions, deletions or single base changes which are more appropriate as substrates for cleavage34. This may suggest a significant degree of heterogeneity at the target site in conjunction with point mutation repair or perhaps independent of it. A similar observation was made by Schumann et al. using a T7 Endonuclease I (T7E1) recognition assay to measure CRISPR/Cas9 cleavage activity37. In that system, however, the objective was different; to insert a segment of DNA.


Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides
Correlation between CRISPR/Cas9 cleavage and gene editing activity.Synchronized and released HCT116-19 cells were electroporated with 0.0–10.0 μg of pX330 and with (+ODN) or without (−ODN) 1.35 μg of 72-mer. CRISPR/Cas9 cleavage activity measured by Surveyor endonuclease assay (orange and yellow) as well as gene editing (dark green and light green) activity measured by FACS are shown. Standard error is represented by the bars on the each data point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016854&req=5

f3: Correlation between CRISPR/Cas9 cleavage and gene editing activity.Synchronized and released HCT116-19 cells were electroporated with 0.0–10.0 μg of pX330 and with (+ODN) or without (−ODN) 1.35 μg of 72-mer. CRISPR/Cas9 cleavage activity measured by Surveyor endonuclease assay (orange and yellow) as well as gene editing (dark green and light green) activity measured by FACS are shown. Standard error is represented by the bars on the each data point.
Mentions: Next, DNA cleavage activity, generated through the action of the CRISPR/Cas9 system, was determined by the Surveyor endonuclease at dosages that support significant levels of gene editing. The data are presented in Fig. 3 and supplementary Figure S1. The Surveyor assay successfully detected CRISPR/Cas9 activity in the absence of the ssODN; a predictable rise in activity was observed as a function of the amount of expression vector present in the reaction. When cleavage activity is measured in complete reaction mixtures, i. e. in the presence of the oligonucleotide, Surveyor endonuclease activity is increased (orange line vs yellow line), particularly at the two doses where gene editing activity is near maximal. While this may appear to be counterintuitive, the Surveyor endonuclease assay has a preference for DNA duplexes with small but definitive indels. DNA duplexes bear small insertions, deletions or single base changes which are more appropriate as substrates for cleavage34. This may suggest a significant degree of heterogeneity at the target site in conjunction with point mutation repair or perhaps independent of it. A similar observation was made by Schumann et al. using a T7 Endonuclease I (T7E1) recognition assay to measure CRISPR/Cas9 cleavage activity37. In that system, however, the objective was different; to insert a segment of DNA.

View Article: PubMed Central - PubMed

ABSTRACT

The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

No MeSH data available.


Related in: MedlinePlus