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. 2018 Oct 17;19(1):164.
doi: 10.1186/s13059-018-1532-z.

CRISPR-induced exon skipping is dependent on premature termination codon mutations

Tingting Sui  1 , Yuning Song  1 , Zhiquan Liu  1 , Mao Chen  1 , Jichao Deng  1 , Yuanyuan Xu  1 , Liangxue Lai  2   3 , Zhanjun Li  4
Affiliations

CRISPR-induced exon skipping is dependent on premature termination codon mutations

Tingting Sui et al. Genome Biol. .

Abstract

In previous studies, CRISPR/Cas9 was shown to induce unexpected exon skipping; however, the mechanism by which this phenomenon is triggered is controversial. By analyzing 22 gene-edited rabbit lines generated using CRISPR/Cas9, we provide evidence of exon skipping at high frequency in premature termination codon-mutated rabbits but not in the rabbits with a premature termination codon mutation in exon 1 rabbits with non-frameshift or missense mutations. Our results suggest that CRISPR-mediated exon skipping depends on premature termination codon mutation-induced nonsense-associated altered splicing.

Keywords: CRISPR/Cas9; Exon skipping; PTC; Rabbits.

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Conflict of interest statement

Ethics approval and consent to participate

The gene editing rabbits were generated in our group, all procedures using rabbits were approved by the Animal Care and Use Committee of Jilin University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Exon skipping induced using the CRISPR/Cas9 system. a No exon skipping in rabbits with a non-frameshift mutation. b Non-frame shift mutation in exon 6 of GCK did not induce exon skipping. Schematic diagram of sgRNA target site in exon 3 of the rabbit GCK gene locus and RT-PCR analysis of GCK gene-editing rabbits for exons 2, 3, 4 and 5. Gel images have been cropped. M, which shows the DL2000 ladder, indicates band size. K1, K2, K3, the GCK gene-edited rabbits used in this study. c CRISPR-mediated exon skipping depends on PTC mutation-induced nonsense-associated altered splicing (NAS). d PTC mutation in exon 12 of ANO5 gene induces exon skipping. Schematic of sgRNA target site in exon 12 of the rabbit ANO5 gene and RT-PCR analysis of ANO5 gene-editing rabbits for exons 10, 11, 12, 13 and 14. Gel images have been cropped. M, which shows the DL2000 ladder, indicates band size. A1-A2, the ANO5 gene-edited rabbits used in this study. e No exon skipping in mutated rabbits with a PTC in exon 1. Rectangle, exon; blue octagon, normal stop codon; red octagon, PTC; NMD, nonsense-mediated decay; NAS, nonsense-associated alternative splicing; ATG, initiation codon; E1-E5, different exons. (F) PTCs mutation in exon 1 of MSTN gene did not induce exon skipping. Schematic diagram of sgRNA target site in exon 1 of the rabbit MSTN gene locus and RT-PCR analysis of MSTN gene editing rabbits for exons 1, 2 and 3. Gel images have been cropped. M, which shows the DL2000 ladder, indicates band size. M1, M2, M3, the MSTN gene-edited rabbits used in this study
See this image and copyright information in PMC

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