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. 2018 Aug 15;19(1):107.
doi: 10.1186/s13059-018-1482-5.

CRISPR-SKIP: Programmable Gene Splicing With Single Base Editors

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Free PMC article

CRISPR-SKIP: Programmable Gene Splicing With Single Base Editors

Michael Gapinske et al. Genome Biol. .
Free PMC article

Abstract

CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes through targeted double-strand breaks in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for off-target mutations, technologies capable of introducing targeted changes with increased precision, such as single-base editors, are preferred. We present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.

Keywords: Alternative splicing; BRCA2; Base editing; CRISPR-Cas9; Exon skipping; Gene editing; Gene isoform; PIK3CA; RELA; Synthetic biology.

Conflict of interest statement

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
CRISPR-SKIP targeting strategy. a The consensus sequence of splice acceptors. We hypothesize that base editing of the highly conserved G (asterisk) leads to exon skipping. b In the presence of an appropriate PAM sequence, base editors can be utilized to deaminate the cytidine in the antisense strand, which is complementary to the conserved guanosine in the splice acceptor, thus resulting in the disruption of the splice acceptor and exon skipping
Fig. 2
Fig. 2
Single-base editing of splice acceptor consensus sequences enables programmable exon skipping. a 293T cells were transfected with C>T base editors and sgRNAs targeting the splice acceptor of exon 7 in RELA. RT-PCR was used to detect exon skipping over a 10-day time course. b Skipping of RELA exon 7 and PIK3CA exon 5 was induced by C>T base editors, but not by the sgRNA alone or in combination with dead SpCas9 or D10A nickase SpCas9. c Sanger sequencing of the exon-skipped amplicon was used to demonstrate successful exon skipping of RELA exon 7 and PIK3CA exon 5. d Deep sequencing of genomic DNA in wild-type (WT) cells and cells treated with C>T base editors targeting RELA exon 7 and PIK3CA exon 5 was used to calculate the modification rate. e Quantification of the rate of exon skipping of RELA exon 7 and PIK3CA exon 5 by deep sequencing of mature mRNA, which was amplified by RT-PCR
Fig. 3
Fig. 3
CRISPR-SKIP is effective across a panel of cell lines. CRISPR-SKIP induced skipping of RELA exon 7 and PIK3CA exon 5 in the cell lines HCT116, HEPG2, and MCF7
Fig. 4
Fig. 4
Comparison of CRISPR-SKIP with active SpCas9 for inducing exon skipping. CRISPR-SKIP was utilized to target the splice acceptors of RELA exon 7, PIK3CA exon 5, and JAG1 exon 9. In parallel, sgRNAs targeting the same exons were co-transfected with active SpCas9 to induce exon skipping. Analysis by PCR demonstrates that CRISPR-SKIP induced exon skipping at equal or greater rates than active SpCas9 in each of three exons tested
Fig. 5
Fig. 5
Different Cas9 scaffolds increase the number of CRISPR-SKIP target exons. a, b RT-PCR analysis demonstrates that SpCas9-VQR-BE3 (a) and SaCas9-KKH-BE3 (b) can induce exon skipping of BRCA2 exon 26 and RELA exon 10, respectively. c, d Deep sequencing of genomic DNA revealed that targeted mutations (red) introduced by SpCas9-VQR-BE3 were found in 0.93% of reads at the BRCA2 exon 26 splice acceptor (c), while SaCas9-KKH-BE3 induced targeted mutations in 46.61% of reads at RELA exon 10 splice acceptor (d). Deep sequencing was performed in biological duplicates, and the results were combined. e, f Quantification of the rate of exon skipping of BRCA2 exon 26 (e) and RELA exon 10 (f) by deep sequencing of mature mRNA, which was amplified by RT-PCR. RNAseq was performed on biological duplicates and a single estimate of the proportion and confidence intervals were obtained (“Methods”)
Fig. 6
Fig. 6
CRISPR-SKIP can be used to simultaneously skip multiple exons within the same transcript. SaCas9-KKH-BE3 was used to target PIK3CA exons 11 and 12. RT-PCR demonstrated that both sgRNAs induced skipping of the targeted exon and, when used together, induced skipping of both exons simultaneously
Fig. 7
Fig. 7
Genome-wide computational estimation of targetability by CRISPR-SKIP. a Estimation of the number of exons that can be targeted by each base editor with estimated efficiency of editing flanking intronic G at or above the corresponding value on the x-axis. Only exons with maximum off-target score below 10 are considered. b Estimation of the number of exons that can be targeted by each base editor with maximum off-target score at or below the corresponding value on the x-axis. Only exons for which the estimated efficiency of editing the flanking G nucleotide is above 20% are considered

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