During CRISPR-directed gene editing, multiple gene repair mechanisms interact to produce a wide and largely unpredictable variety of sequence changes across an edited population of cells. Shortcomings inherent to previously available proposal-based insertion and deletion (indel) analysis software necessitated the development of a more comprehensive tool that could detect a larger range and variety of indels while maintaining the ease of use of tools currently available. To that end, we developed De convolution of Co mplex D NA R epair (DECODR). DECODR can detect indels formed from single or multi-guide CRISPR experiments without a limit on indel size. The software is accurate in determining the identities and positions of inserted and deleted bases in DNA extracts from both clonally expanded and bulk cell populations. The accurate identification and output of any potential indel allows for DECODR analysis to be executed in experiments utilizing potentially any configuration of donor DNA sequences, CRISPR-Cas, and endogenous DNA repair pathways.

muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous formuREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2′-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.

Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.