Target Site Mutagenesis during Crispr/ Cas 9/Single-Stranded- Oligonucleotide Directed Gene Editing for Sickle Cell Anemia

Introduction

Sickle Cell Disease (SCD) results from a simple substitution of valine for glutamic acid at codon 6 in the β globin gene, resulting in an AàT transition in the third position. The mutation results in the production of hemoglobin HbS which differs from the normal HbAin that it tends to polymerize into long strands that deform the erythrocyte. While a variety of traditional treatment regimens or reagents, such as hydroxyurea and chronic transfusions have been used widely, these therapies are wrought with short and long term side effects that limit efficacy. There is great interest in the hypothesis that the repair of a single nucleotide is facilitated by the combined action of CRISPR/Cas 9 and single-stranded oligonucleotides (ssODNs) could prove a significant therapeutic advance for sickle cell disease. CRISPR/Cas 9 induces a site-specific double-stranded break while the single-stranded oligonucleotide provides a DNA template to improve the rate of accurate genetic correction. There is a great effort to understand off-site mutagenesis caused by editing of DNA regions remote to the target sequence. It is critical that investigators understand the frequency and types of DNA alterations induced by the gene editing reaction and affecting the region surrounding the targeted nucleotide site (herein termed on-site mutagenesis).

Methods

We targeted beta globin genes with a CRISPR/Cas9 system designed to cleave 2 bases to the 5'side of the targeted (A) nucleotide (the PAM site is located 2 bases to the 3' side on the complimentary strand) coupled to the electroporation of a single-stranded oligonucleotide (72-mer) designed to convert the wild-type (A) to the mutant (T) nucleotide. To evaluate the rate of on-site mutagenesis among individual alleles, we clonally expanded populations of edited K562 cells. We hypothesize that the evaluation of individual clones will permit a clear identification of intended and un-intended on-site DNA alteration. Allelic heterogeneity is analyzed using Tracking of Indels by DEcompositon (TIDE) methodology combined with Sanger sequencing.

Results

We isolated 26 clonal lines for continued expansion after evidence of CRISPR/Cas 9 plasmid uptake and activity. Sanger sequencing with TIDE analysis revealed that the DNA sequence surrounding the targeted base is altered significantly as a result of the CRISPR/Cas9 gene editing process. Twenty three percent of the clones contain at least one corrected allele but one hundred percent of the clones exhibited mutagenicity of the DNA sequence surrounding the targeted base. All clones analyzed displayed varying degrees of sequence alteration (Figure 1). Interestingly, one clone contained a DNA insertion homologous to a region of the delta globin gene, suggesting that gene editing of the beta globin gene may have been repaired, in part, by delta globin DNA. The sequence of the delta globin locus was not changed.

Conclusions

Taken together, our data suggest that combinatorial approaches to beta globin gene editing using CRISPR/Cas 9 and single-stranded oligonucleotides induce significant onsite mutagenesis and potential genetic swapping between related members of the same gene family. These observations provide insight into the type of molecular activity that accompanies combinatorial gene editing, particularly surrounding the target site.

This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM109021

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