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, 10 (6), e0129308
eCollection

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

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Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

Pawel Bialk et al. PLoS One.

Abstract

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Experimental Design and Mutant eGFP Gene Editing System (A) CRISPR/Cas9 constructs were designed and built following published guidelines and protocols [42].
Unsynchronized or synchronized and released cells were transfected via electroporation with CRISPR/Cas9 construct and ssODNs and allowed to recover before analysis. Gene editing was assessed by flow cytometry and CRISPR/Cas9 activity was measured by RFLP using restriction enzyme AvrII. (B) A segment of the mutant eGFP gene is shown with the three ssODNs, 72NT, 72T and 72NT PM respectively aligned above or below their respective binding sites. Phosphorothioate modified and protected ends are denoted with *.The five arbitrarily named (1C-5C) custom CRISPR/Cas9s RNA guide sequences are depicted in blue with their predicted cleavage sites shown by the red arrows. The effected codon is shown in bold with the mutant base to be edited in red. The base driving the gene editing conversion is shown in green.
Fig 2
Fig 2. Summary of Constructed CRISPR/Cas9 gRNAs.
Each designed guide RNA was generated from the crispr.mit.edu online algorithm. CRISPR # designates the gRNA name used in this manuscript. Score represents the likelihood of the gRNA binding and causing unwanted mutations (score of 100 is the best possible gRNA). Possible off-target effects is the total number of individual loci across the genome that could be cleaved with the number of those within in genes (exons) listed in the following column. RNA guide binding strand denotes which stand the guide RNA will target (NT = non-transcribed, T = transcribed). Cut site is where the gRNA will direct the DSB break to be made relative to TAG = 0. CRISPR binding element shows the segment of the eGFP gene that the guide will bind with the actual bound strand in bold. Oligos used for construction with correct linkages are shown in the final column.
Fig 3
Fig 3. Free Energy Heterodimer Values for CRISPR Guide RNA and ssODN Combinations.
Each gRNA sequence was aligned and analyzed for base pairing and maximum free energy (ΔG) values utilizing the IDT heterodimer calculator and measured in kcals/mole. The highest ΔG value for each gRNA/72NT ssODN pairing is shown. Solid lines represent the longest stretch of direct base pairing while dotted lines represent additional complimentary bases (not calculated in ΔG). A more negative ΔG value represents a stronger binding capacity.
Fig 4
Fig 4. CRISPR/Cas9 and ssODN Gene Editing Activity.
Unsynchronized HCT116-19 cells were harvested and electroporated at a concentration of 5x105cells/100ul with 2ug of the indicated CRISPR/Cas9 plus 1.35ug of either 72NT or 72T. Following electroporation, cells were allowed to incubate for 48 hours and correction efficiency was determined by the percentage of total viable eGFP+ cells in the population. 1C– 5C CRISPR/Cas9 complexes and are listed left to right relative to their cut site. The numbers below indicate the average correction efficiency. Error bars represent standard error.
Fig 5
Fig 5. CRISPR/Cas9 Cleavage Activity.
(A) 2% TBE agarose gel analysis of cleavage products generated by 2C and 3C CRISPR/Cas9 complexes at the eGFP gene target site. AvrII restriction enzyme was used to digest the amplified region of the eGFP gene. 2C/-, CRISPR 2C with no ssODN; 2C/+, CRISPR 2C with 72NT etc. Untreated 181bp (U) and Untreated + AvrII 181bp PCR products were used as internal controls with AvrII digested 2C, 3C and 2C + 72NT PM and 3C 72NT PM 181bp PCR samples. Densitometry was performed on all samples and percent cleavage (181bp band) is indicated below each sample. (B) SURVEYOR assay comparing the cleavage efficacy of each gRNA as the percent of indel formation. Arrowheads indicate parental bands and cleaved products.
Fig 6
Fig 6. Gene Editing Activity with CRISPR/Cas9 Nickases and ssODNs.
Unsynchronized HCT116-19 cells were electroporated at a concentration of 5x105 cells/100ul with 2ug of the indicated CRISPR/Cas9 Nickase (1N, 2N, 3N, 4N, 5N) plus 1.35ug of 72NT. Following electroporation, cells were allowed to incubate for 48 hours. Correction efficiency was determined by the percentage of total viable eGFP+ cells in the population as described previously. Each treatment was performed in duplicate and error bars represent standard error.
Fig 7
Fig 7. Double Nicking Nuclease Array of Gene Editing.
Unsynchronized HCT116-19 cells were electroporated with 1ug of each of the indicated combinations of CRISPR/Cas9 nickases (1N, 2N, 3N, 4N, 5N) plus 1.35ug of 72NT. Offsets denoted with a star (*) represent nicking pairs which induce nicks on the same strand. Following electroporation, cells were allowed to incubate for 48 hours. Correction efficiency was determined by the percentage of total viable eGFP+ cells in the population as described previously. Each treatment was performed in duplicate and error bars represent standard error.
Fig 8
Fig 8. Activity Profile of Gene Editing catalyzed by TALENs, CRISPRs or Nickases at the target eGFP gene.
ssODN directed gene editing activity utilizing TALENs, CRISPR/Cas9s or CRISRP/Cas9 nickases was compiled and plotted within the region of the target eGFP gene. Cytosine of the corrected tyrosine codon is designated as base 0. TALEN data was derived from previous work [15]. tatistical analysis was performed using two-sample unequal variance students T-test distribution. *p<0.05 (see Table 2).
Fig 9
Fig 9. Sequence confirmation of ssODN/CRISPR edited cells.
Unsynchronized HCT116-19 cells were electroporated under the following conditions; 2ug CRISPR 2C and 1.35ug 72NT at 5e5 cells/100ul. Cells were then sorted for GFP+ at 72 hours post electroporation. Immediately following cell sorting, DNA was isolated and the region surrounding the target base was amplified via PCR. Samples were submitted to Genewiz (South Plainfield, NJ) for sequencing analysis.

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