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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.

No MeSH data available.


(A) Experimental workflow. HCT116-19 cells are either unsynchronized or synchronized and released, then transfected with a CRISPR/Cas9 expression vector (pX330) with or without ssODN, then after 48 hours analyzed for gene editing activity and Surveyor endonuclease digestion. (B) Gene editing model system and ssODNs. The wild-type and mutated eGFP gene segments with the target codon located in the center of the sequences are displayed in green and red, respectively. The nucleotide targeted for exchange is emphasized in bold and underlined. 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 which is used to target the mutated eGFP gene is shown.
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f1: (A) Experimental workflow. HCT116-19 cells are either unsynchronized or synchronized and released, then transfected with a CRISPR/Cas9 expression vector (pX330) with or without ssODN, then after 48 hours analyzed for gene editing activity and Surveyor endonuclease digestion. (B) Gene editing model system and ssODNs. The wild-type and mutated eGFP gene segments with the target codon located in the center of the sequences are displayed in green and red, respectively. The nucleotide targeted for exchange is emphasized in bold and underlined. 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 which is used to target the mutated eGFP gene is shown.

Mentions: The repair of a single point mutation by gene editing can be evaluated using a well-established reporter gene system consisting of a single copy of a mutant eGFP gene integrated into HCT116 cells111530. Repair of this mutation is executed by the combined action of a specifically designed ssODN, 72 bases in length, and the appropriate CRISPR/Cas9 system17. The cells can be targeted as either an unsynchronized population or synchronized and released population, as shown in Fig. 1A. The reaction is initialized through strand invasion by the ssODN and subsequent alignment in homologous register with the target site (here, the mutant eGFP gene) except for a single base pair mismatch located in the center33. Figure 1B illustrates the alignment of the ssODN with the appropriate strand of the mutant eGFP gene. Here, we evaluated gene editing activity by the combined action of the ssODN, and a CRISPR/Cas9 system previously designed for the same target1117 and concurrently measured CRISPR/Cas9 cleavage activity using the well-known Surveyor endonuclease assay34. Since this is a reporter gene, off-target effects are minimized, yet we include the ten top predicted off target sites in supplementary Table S1.


Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides
(A) Experimental workflow. HCT116-19 cells are either unsynchronized or synchronized and released, then transfected with a CRISPR/Cas9 expression vector (pX330) with or without ssODN, then after 48 hours analyzed for gene editing activity and Surveyor endonuclease digestion. (B) Gene editing model system and ssODNs. The wild-type and mutated eGFP gene segments with the target codon located in the center of the sequences are displayed in green and red, respectively. The nucleotide targeted for exchange is emphasized in bold and underlined. 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 which is used to target the mutated eGFP gene is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: (A) Experimental workflow. HCT116-19 cells are either unsynchronized or synchronized and released, then transfected with a CRISPR/Cas9 expression vector (pX330) with or without ssODN, then after 48 hours analyzed for gene editing activity and Surveyor endonuclease digestion. (B) Gene editing model system and ssODNs. The wild-type and mutated eGFP gene segments with the target codon located in the center of the sequences are displayed in green and red, respectively. The nucleotide targeted for exchange is emphasized in bold and underlined. 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 which is used to target the mutated eGFP gene is shown.
Mentions: The repair of a single point mutation by gene editing can be evaluated using a well-established reporter gene system consisting of a single copy of a mutant eGFP gene integrated into HCT116 cells111530. Repair of this mutation is executed by the combined action of a specifically designed ssODN, 72 bases in length, and the appropriate CRISPR/Cas9 system17. The cells can be targeted as either an unsynchronized population or synchronized and released population, as shown in Fig. 1A. The reaction is initialized through strand invasion by the ssODN and subsequent alignment in homologous register with the target site (here, the mutant eGFP gene) except for a single base pair mismatch located in the center33. Figure 1B illustrates the alignment of the ssODN with the appropriate strand of the mutant eGFP gene. Here, we evaluated gene editing activity by the combined action of the ssODN, and a CRISPR/Cas9 system previously designed for the same target1117 and concurrently measured CRISPR/Cas9 cleavage activity using the well-known Surveyor endonuclease assay34. Since this is a reporter gene, off-target effects are minimized, yet we include the ten top predicted off target sites in supplementary Table S1.

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.