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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

HBB gene editing experimental flow.The K562 cells were transfected with the CRISPR/Cas9 and ssODN followed by a 72 hour incubation period. Targeted cells exhibiting eGFP expression were sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. DNA was then isolated and the HBB gene was amplified and subjected to Sanger sequencing and TIDE analyses to investigate the gene editing activity around the target site of the clonally expanded populations.
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f5: HBB gene editing experimental flow.The K562 cells were transfected with the CRISPR/Cas9 and ssODN followed by a 72 hour incubation period. Targeted cells exhibiting eGFP expression were sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. DNA was then isolated and the HBB gene was amplified and subjected to Sanger sequencing and TIDE analyses to investigate the gene editing activity around the target site of the clonally expanded populations.

Mentions: A CRISPR/Cas9 complex that induces DNA cleavage at the indicated site (Fig. 4) was chosen because it had the lowest potential for off-target activity yet would still induce a DNA break site closest to the target nucleotide in the beta globin gene. We also depict the 72-mer oligonucleotide that has been designed specifically to create a mismatch within the adenosine nucleotide (A, in bold) within the wild-type beta-globin (HBB) gene. Resolution of this point mutation through gene editing results in the creation of a T at the mismatch site and subsequently a T:A base pair at the target site (illustrated in Red). The CRISPR/Cas9 targeting system is harbored in an expression vector which also contains a wild-type eGFP gene so that K562 cells successfully transfected are discernible by FACS. The overall experimental flow, as illustrated in Fig. 5, was designed with the ultimate goal of full-scale analyses of sequence alterations. The cells exhibiting eGFP expression were sorted using a FACS Aria II and placed into individual wells for single cell clonal expansion. Cells were allowed to expand for approximately 3–5 weeks at which time DNA was extracted from the clonal expansions and prepared for sequence analysis. Figure 5 (lower panels) displays the results of the Sanger sequencing of 1 individual/expanded clonal population (Clone 22) as well as Sanger sequencing trace files analyzed by the software program, Tracking of Indels by decomposition (TIDE)43. The TIDE program facilitates the examination of individual allelic sequences within complex Sanger sequencing data, which often displays a multiplicity of nucleotide peaks at or surrounding the position of DSB repair. An algorithm that isolates individual sub-sequences followed by an alignment mechanism relative to the control parental sequence to determine the indel structure of individual alleles is utilized. In addition, TIDE affords the opportunity for qualitative analysis of clonally expanded populations, analyses that can provide unique insight into the cleavage profile of each allele in individual targeted cells. As an example of our analytical strategy, we display data from Clone 22 which harbors homozygosity for the biallelic A to T conversion, as indicated by the nucleotides presented in red. We know that while point mutation repair has occurred, each allele contains a different indel; the TIDE indel distribution displays a 2 base deletion (allele 1) and a 1 base insertion (allele 2).


Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides
HBB gene editing experimental flow.The K562 cells were transfected with the CRISPR/Cas9 and ssODN followed by a 72 hour incubation period. Targeted cells exhibiting eGFP expression were sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. DNA was then isolated and the HBB gene was amplified and subjected to Sanger sequencing and TIDE analyses to investigate the gene editing activity around the target site of the clonally expanded populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: HBB gene editing experimental flow.The K562 cells were transfected with the CRISPR/Cas9 and ssODN followed by a 72 hour incubation period. Targeted cells exhibiting eGFP expression were sorted using a FACSAria II flow cytometer as single cells into individual wells for clonal expansion. DNA was then isolated and the HBB gene was amplified and subjected to Sanger sequencing and TIDE analyses to investigate the gene editing activity around the target site of the clonally expanded populations.
Mentions: A CRISPR/Cas9 complex that induces DNA cleavage at the indicated site (Fig. 4) was chosen because it had the lowest potential for off-target activity yet would still induce a DNA break site closest to the target nucleotide in the beta globin gene. We also depict the 72-mer oligonucleotide that has been designed specifically to create a mismatch within the adenosine nucleotide (A, in bold) within the wild-type beta-globin (HBB) gene. Resolution of this point mutation through gene editing results in the creation of a T at the mismatch site and subsequently a T:A base pair at the target site (illustrated in Red). The CRISPR/Cas9 targeting system is harbored in an expression vector which also contains a wild-type eGFP gene so that K562 cells successfully transfected are discernible by FACS. The overall experimental flow, as illustrated in Fig. 5, was designed with the ultimate goal of full-scale analyses of sequence alterations. The cells exhibiting eGFP expression were sorted using a FACS Aria II and placed into individual wells for single cell clonal expansion. Cells were allowed to expand for approximately 3–5 weeks at which time DNA was extracted from the clonal expansions and prepared for sequence analysis. Figure 5 (lower panels) displays the results of the Sanger sequencing of 1 individual/expanded clonal population (Clone 22) as well as Sanger sequencing trace files analyzed by the software program, Tracking of Indels by decomposition (TIDE)43. The TIDE program facilitates the examination of individual allelic sequences within complex Sanger sequencing data, which often displays a multiplicity of nucleotide peaks at or surrounding the position of DSB repair. An algorithm that isolates individual sub-sequences followed by an alignment mechanism relative to the control parental sequence to determine the indel structure of individual alleles is utilized. In addition, TIDE affords the opportunity for qualitative analysis of clonally expanded populations, analyses that can provide unique insight into the cleavage profile of each allele in individual targeted cells. As an example of our analytical strategy, we display data from Clone 22 which harbors homozygosity for the biallelic A to T conversion, as indicated by the nucleotides presented in red. We know that while point mutation repair has occurred, each allele contains a different indel; the TIDE indel distribution displays a 2 base deletion (allele 1) and a 1 base insertion (allele 2).

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