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

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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

Experimental strategy for targeting the human HBB gene.The wild-type and Sickle Cell HBB gene sequences are shown with the single nucleotide mutation characteristic of Sickle Cell Disease emphasized in red. The gRNA and protospacer adjacent motif (PAM) shown indicate the CRISPR/Cas9 target site and the location of the resulting double-stranded break (DSB). The phosphorothioate modified, end protected 72-mer is used to create a mismatch at the mutation site indicated in bold.
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f4: Experimental strategy for targeting the human HBB gene.The wild-type and Sickle Cell HBB gene sequences are shown with the single nucleotide mutation characteristic of Sickle Cell Disease emphasized in red. The gRNA and protospacer adjacent motif (PAM) shown indicate the CRISPR/Cas9 target site and the location of the resulting double-stranded break (DSB). The phosphorothioate modified, end protected 72-mer is used to create a mismatch at the mutation site indicated in bold.

Mentions: The correlation between the extent of single base gene editing and the degree of heterogeneity detected by the Surveyor assay prompted us to examine the DNA sequence both at and surrounding the target site, as a function of point mutation repair. A testable hypothesis is that point mutation repair could be accompanied by various degrees of onsite mutagenesis, resulting in a population of heterogeneous re-ligated DNA ends, the resection products. In order for our observations and data to have more relevance, we decided to do a detailed analysis of single base repair, as it may relate to onsite mutagenesis, on a native gene target. The beta globin gene in K562 cells is a well-established and well-known target for gene editing particularly with regard to Sickle Cell Disease383940. A mutation in the sixth codon of the beta globin gene is responsible for the onset of Sickle Cell Disease and, as such, a reversal of that point mutation through gene editing would have immediate translational applications. We utilized K562 cells which harbor 2–3 copies of the normal beta globin gene4142, bearing a wild type nucleotide (A), at position 16 in the coding region (GAG), as displayed in Fig. 4. We employed the same strategy used for eGFP gene editing; a CRISPR/Cas9 system and the appropriate single-stranded oligonucleotide to convert the A to a T nucleotide.


Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides
Experimental strategy for targeting the human HBB gene.The wild-type and Sickle Cell HBB gene sequences are shown with the single nucleotide mutation characteristic of Sickle Cell Disease emphasized in red. The gRNA and protospacer adjacent motif (PAM) shown indicate the CRISPR/Cas9 target site and the location of the resulting double-stranded break (DSB). The phosphorothioate modified, end protected 72-mer is used to create a mismatch at the mutation site indicated in bold.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Experimental strategy for targeting the human HBB gene.The wild-type and Sickle Cell HBB gene sequences are shown with the single nucleotide mutation characteristic of Sickle Cell Disease emphasized in red. The gRNA and protospacer adjacent motif (PAM) shown indicate the CRISPR/Cas9 target site and the location of the resulting double-stranded break (DSB). The phosphorothioate modified, end protected 72-mer is used to create a mismatch at the mutation site indicated in bold.
Mentions: The correlation between the extent of single base gene editing and the degree of heterogeneity detected by the Surveyor assay prompted us to examine the DNA sequence both at and surrounding the target site, as a function of point mutation repair. A testable hypothesis is that point mutation repair could be accompanied by various degrees of onsite mutagenesis, resulting in a population of heterogeneous re-ligated DNA ends, the resection products. In order for our observations and data to have more relevance, we decided to do a detailed analysis of single base repair, as it may relate to onsite mutagenesis, on a native gene target. The beta globin gene in K562 cells is a well-established and well-known target for gene editing particularly with regard to Sickle Cell Disease383940. A mutation in the sixth codon of the beta globin gene is responsible for the onset of Sickle Cell Disease and, as such, a reversal of that point mutation through gene editing would have immediate translational applications. We utilized K562 cells which harbor 2–3 copies of the normal beta globin gene4142, bearing a wild type nucleotide (A), at position 16 in the coding region (GAG), as displayed in Fig. 4. We employed the same strategy used for eGFP gene editing; a CRISPR/Cas9 system and the appropriate single-stranded oligonucleotide to convert the A to a T nucleotide.

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