Frameshift indels introduced by genome editing can lead to in-frame exon skipping

<div><p>The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of “multiple of three nucleotides” exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a “multiple of three nucleotides” exon of <i>LGALS8</i> that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.</p></div

Exon skipping in <i>LGALS8</i> in human erythroblasts.

<p>(<b>A</b>) In <i>in vitro</i> differentiated human erythroblasts, three <i>LGALS8</i> mRNA isoforms are expressed. Isoform 1 includes the “multiple of three nucleotides” exon 9 (in red, 126-bp), whereas isoforms 2 and 3 do not. The nonsense variant rs2273865 (p.Leu212Ter) is located in exon 9. At this variant, the minor A-allele has a frequency of 3.5% in populations of European ancestry (ExAC). (<b>B</b>) Eight erythroblast samples are heterozygous at rs2273865 and show strong allelic imbalance (binomial <i>P</i><0.05 for all samples). Numbers in the bars indicate the numbers of reads carrying the T (green) or A (blue) allele. Differential expression of total <i>LGALS8</i> (<b>C</b>), isoform 1 (<b>D</b>), isoform 2 (<b>E</b>), and isoform 3 (<b>F</b>) between erythroblast samples homozygous TT (n = 16) and heterozygous AT (n = 8) at rs2273865. No samples homozygous for the minor allele (AA) were available. (<b>G</b>) The ratio of <i>LGALS8</i> transcripts without exon 9 over transcripts with exon 9 is higher in heterozygous AT than in homozygous TT erythroblast samples. <i>P</i>-values are calculated by linear regression correcting for cell developmental stage.</p

Frameshift indels cause in-frame exon skipping in <i>PHACTR1</i>.

<p>(<b>A</b>) <i>PHACTR1</i> expression levels measured by real-time qPCR in the parental teloHAEC cell line, an unedited clone (sg-E8N23), and clones with CRISPR-Cas9-generated frameshift indels in <i>PHACTR1</i> exon 8 (sg-E8N2 and sg-E8N16), exon 9 (sg-E9N1), and exon 10 (sg-E10N8). Data show mean and standard error of the mean from two biological replicates, done in triplicates. <i>PHACTR1</i> expression levels in sg-E8N2 is 6.2 fold greater than in the parental teloHAEC cell line (Student’s <i>t</i>-test <i>P</i> = 0.0033). (<b>B</b>) Agarose gel electrophoresis profile of the main <i>PHACTR1</i> isoforms detected in cDNA from teloHAEC cells, unedited clones, or clones with a frameshift indel. We assigned a transcript number to each of the <i>PHACTR1</i> isoform that we could Sanger sequence and align to the reference sequence. Unlabeled bands could not be assigned to <i>PHACTR1</i>. Bands in the molecular ladder correspond to 400, 500 and 700-bp. This gel is representative of three independent experiments. (<b>C</b>) Schematic diagram of all the <i>PHACTR1</i> isoforms that we identified in the different teloHAEC cell lines. Transcript numbers correspond to the bands (white numbers) in <b>B</b>. The PCR primers in exon 6 and 11 are depicted. For the isoforms expressed in edited clones, we added the corresponding nucleotide changes introduced by the frameshift indels. (<b>D</b>) Western blot of PHACTR1 in the parental, unedited and edited teloHAEC cells. The arrowhead indicates PHACTR1, lower bands are non-specific proteins recognized by the antibody. PHACTR1 is smaller in sg-E8N2, consistent with skipping of exon 8 or usage of an alternative in-frame start codon downstream of the frameshift indel. For sg-E9N1, the smaller PHACTR1 protein is consistent with skipping of exon 9. We could not detect PHACTR1 proteins in sg-E8N16 and sg-E10N8. We used GAPDH as loading control. This Western blot is representative of three independent experiments.</p

Frameshift indel can lead to exon skipping in the zebrafish <i>adgrl4</i> gene.

<p>(<b>A</b>) <i>adgrl4</i> genomic locus, TALEN target site in exon 2 of the <i>adgrl4</i> gene and a stable mutant line (Δ5) that was analyzed. (<b>B</b>) PCR analysis of <i>adgrl4</i> mRNA transcripts in 10 to 15 pooled embryo samples from control (ctrl), <i>adgrl4</i> Δ5^+/-, and <i>adgrl4</i> Δ5^-/- fishes. (<b>C</b>) Schematic representation of the different transcripts recovered from the bands (white numbers) in <b>B</b>.</p

Flowchart illustrating the "n" values between groups throughout the study.

<p>Flowchart illustrating the "n" values between groups throughout the study.</p

Increased ANGPTL2 in post-ACS patients.

<p>Fasting ANGPTL2 levels in the plasma of patients with post-acute coronary syndrome (ACS) (n = 33) compared to age-matched (n = 20) and young (n = 20) healthy controls. Data are mean ± SEM of (n) participants, *: p<0.05 <i>vs</i> Age-matched controls (Kruskal-Wallis test).</p

Detectable <i>ANGPTL2</i> methylation profile.

<p>Quantification of various methylation sites located in the <i>ANGPTL2</i> gene identified following a preliminary genome-wide exploratory assay. DNA samples were pooled from a small number of participants taken from all three groups. Data are mean ± SEM of a total of 16 patients from the young controls (n = 4), age-matched controls (n = 7) and post-ACS patients (n = 5).</p

Flowchart illustrating the "n" values between groups throughout the study.

<p>Flowchart illustrating the "n" values between groups throughout the study.</p

Exploratory probe coordinates.

<p>Genomic localisation of probes covering ANGPTL2 CpG sites analyzed by the Infinium HumanMethylation450 exploratory assay, as provided by the manufacturer.</p

Anthropometric, hemodynamic and metabolic parameters of participants.

<p>Anthropometric, hemodynamic and metabolic parameters of participants.</p