Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements

Abstract

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

Figure 1. Frequency of PigA loss upon editing with exonic and intronic gRNAs in mouse ES cells.

(A) Experimental design. Cells were transfected with separate PiggyBac transposons carrying gRNA and Cas9 genes and selected for stable transposition. PigA negative cells (green) were sorted, single cell clones isolated, the region around the cut site amplified, sequenced and mapped to the reference genome. (B) Examples of PigA editing revealed by FLAER staining, for two gRNAs and one control. (C) Frequency of PigA loss caused by Cas9 with intronic and exonic gRNAs (Table S1; N=6 biologically independent cell cultures). NC = negative control, a guide targeting the Cd9 gene. Thick bars represent exons, hollow ones indicate UTRs.

Figure 2. Analysis of the PigA locus edited with selected gRNAs.

(A) Coverage of PacBio reads at the PigA locus. The locus was PCR amplified from a pool of cells sorted for PigA expression (or from the unsorted population) and the resulting products were sequenced using the PacBio platform. The right panel depicts a 100bp region centered at the cut site. NC: negative control gRNA, ex: exonic gRNA (#56), 5': 5' intronic gRNA (#15), 3': 3' intronic gRNA (#10). The cut site of the gRNA (between 3^rd and 4^th nucleotide from the PAM sequence) is indicated with a vertical black bar. Genomic position is given with respect to the GRCm38 reference genome. N=1. (B-D) Examples of alleles. The bottom diagram represents the PigA reference allele around exon 2, the diagram immediately above shows the structure of the sequenced allele. The top diagram in B shows the genomic Hmgn1 gene structure, note the different scale. Black horizontal line – direct reference match, orange bar – inversion, blue bar – insertion from another part of the genome, black arrowhead – gRNA target site. Grey and orange shadows represent, respectively, direct and inverted match between the reference and the sequenced allele. Lack of shadow at the reference locus represents a deletion in the sequenced allele.

Figure 3. Analysis of Cas9 editing at the autosomal Cd9 locus in mouse ES cells.

Experimental setup is analogous to the PigA experiment in Fig. 1A. A mouse ES cell line derived from an F1 cross between Mus musculus castaneus (CAST) and Mus musculus (BL6) was used. (A) Positions of primer pairs and gRNAs (Tables S1 and S6). Genomic position is given with respect to the GRCm38 reference genome. (B) Examples of Cd9 editing revealed by antibody staining, for two gRNAs and one control (Table S1; N=7 biologically independent cell cultures). (C) PacBio alleles derived from Cd9 positive, mixed (bimodal) and negative, individually sequenced single cell clones, displayed as a pileup. Display conventions as in Fig. 2. N=1. (D) Recombinant alleles. Two of the sequenced single cell clones contained alleles indicative of a cross over event between the homologous chromosomes. Red vertical bars in CAST allele (grey bar) indicate positions of sequence divergence from the BL6 reference genome (black bar), dotted black line indicates missing sequence (deletion), thin black line indicates an intron. LOH – loss of heterozygosity.

Figure 4. Frequency of PIGA loss upon editing with exonic and intronic gRNAs and structure of the recovered alleles in human RPE1 cells.

Cas9-expressing cells were transfected with PiggyBac transposons carrying a gRNA and selected for stable transposition. PIGA negative cells were sorted, single cell clones isolated, the region around the cut site amplified, sequenced and mapped to the reference genome. (A) Examples of PIGA editing revealed by FLAER staining, for two gRNAs and one control. (B) Frequency of PIGA loss caused by Cas9 with intronic and exonic gRNAs (Table S1; N=3 biologically independent cell cultures). Position of the primers with the largest span (6kb) is indicated. (C-E) Recovered alleles. (C) 5' intronic guide #275, (C) 5' intronic guide #274, (D) 3’ intronic guide #276. The position of the gRNA is shown as a vertical line intersecting with the PIGA gene structure. Pure insertions and deletions of <50bp are indicated with orange and black circles, respectively. Combined insertion/deletion events of <50bp and SNPs (‘indels’) are indicated with a red circle. Black lines represent deletions >50bp. Orange bars indicate size of the >50bp insertions (but not their map position). They are centred on the insertion locus or on the associated deletion. Thin, horizontal, dashed line separates clones.