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.
Highlights
The reengineering of mammalian genomes is a powerful genetic approach to both understanding gene function and developing new therapies for inherited diseases
As a search for adjuvants that can enhance the frequency of singlestranded ODN–directed gene editing has proceeded, it was determined that the double-stranded DNA breaks induced prior to the introduction of the single-stranded ODN elevated the overall activity of gene editing between 5 to 10 fold [5,6,7,8]
By the combined action of the CRISPRCas9/Nickase enzyme complex and the single-stranded oligonucleotide, the TAG codon can be converted to TAC, a tyrosine codon that enables the production of functional eGFP
Summary
The reengineering of mammalian genomes is a powerful genetic approach to both understanding gene function and developing new therapies for inherited diseases. As a search for adjuvants that can enhance the frequency of singlestranded ODN–directed gene editing has proceeded, it was determined that the double-stranded DNA breaks induced prior to the introduction of the single-stranded ODN elevated the overall activity of gene editing between 5 to 10 fold [5,6,7,8]. While effective in promoting nucleotide exchange, these agents do not act at specific sites so that multiple non-discriminate cleavage events are likely to be taking place across the genome. Such random mutagenesis has the potential to lead to improper or inaccurate gene function or chromosomal activity. There was a need to identify enzymes or reagents whose doublestranded DNA cleavage activity could be programmed or at least partially controlled to act and enhance gene editing
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