title: Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.
abstract: CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.
authors.affiliation: Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.|Department of Medical Sciences University of Delaware, Newark, Delaware, United States of America.
authors.forename: Natalia
authors.initials: N
authors.lastname: Rivera-Torres
authors.affiliation: Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.|Department of Medical Sciences University of Delaware, Newark, Delaware, United States of America.
authors.forename: Kelly
authors.identifier: http://orcid.org/0000-0002-3130-9460
authors.initials: K
authors.lastname: Banas
authors.affiliation: Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.
authors.forename: Pawel
authors.initials: P
authors.lastname: Bialk
authors.affiliation: Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.|Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware, United States of America.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bloh
authors.affiliation: Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.|Department of Medical Sciences University of Delaware, Newark, Delaware, United States of America.
authors.forename: Eric B
authors.initials: EB
authors.lastname: Kmiec
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002999:Clone Cells; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D005434:Flow Cytometry; D000072669:Gene Editing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D012261:Ribonucleoproteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins