CRISPR-Cas9 technology has been rapidly evolving as a method for gene editing and repair. However, protocols still require procedural improvements that increase editing precision and avoid “collateral mutagenesis” near the target site, and nonspecific editing at unintended genomic sites. In this research report, scientists in Dr Eric Kmiec‘s laboratory demonstrate the advantages of using a CRISPR-Cas9 ribonucleoprotein (RNP) format for DNA cleavage along with single-stranded DNA (ssDNA) oligonucleotides for direct nucleotide exchange at precise positions. The method results in repair of single-base mutations without accompanying insertions and/or deletions. To investigate the mechanism of repair by these gene editing tools, the group examined the genotype of individually sorted, corrected and uncorrected cells after clonal expansion.
For this study, the Kmiec lab used HCT 116 cells containing a single-copy eGFP gene that has a stop codon (TAG) mutation in place of a tyrosine codon (TAC). Using the IDT Alt-R® CRISPR-Cas9 System in conjunction with a repair oligonucleotide, the researchers were able to convert the mutant stop codon to the normal tyrosine. Successful point mutation corrections resulted in functional, detectable eGFP. Following the repair process, the researchers clonally expanded targeted cells with and without a corrected eGFP gene. They then characterized DNA cleavage and repair events, and assessed the cell populations for collateral mutagenesis damage.
The team used genotypic and phenotypic readout of a functional and detectable eGFP to assess point mutation correction. Employing the Alt-R CRISPR-Cas9 System and ssDNA repair oligo resulted in significant targeted gene correction of the mutant base (10–12%, reproducibly).
The authors state that use of an RNP format “delivers the active components of the CRISPR/Cas9 system to the nucleus at approximately the same time, facilitating a more constant initialization of the gene editing reaction.” They assert their studies show that point mutation is repair driven by the combination of the RNP and the ssDNA, as opposed to prior use of a plasmid expression system in which Cas9 is expressed from the same plasmid as the sgRNA.
The group also examined the targeted region of sorted, clonally expanded, cell populations containing the corrected and uncorrected gene (based on eGFP expression) to evaluate any collateral mutagenesis left by the action of the gene editing tools.
DNA sequence analysis of the eGFP expressing cells showed exact point mutation repair, with no adjacent sequence modification. However, similar analysis of cell populations containing an uncorrected gene revealed frequent collateral mutagenesis, with deletions and insertions surrounding the target site. In addition, 2 clonal populations that did not express eGFP did in fact show point mutation repair, but also included collateral mutagenesis near the target site. This latter result led the authors to emphasize the importance of analyzing mutagenicity in uncorrected cells.