CRISPR-SKIP: programmable gene splicing with single base editors

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.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1482-5) contains supplementary material, which is available to authorized users.

Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease

“…Theoretically, multi-exon skipping would be potentially applicable to over 80% of patients with either frame-shift or nonsense mutations in the USH2A gene. Furthermore, exon-skipping could also be achieved by altering the canonical splicing sites of any exon of interest using the latest advanced genetic engineering technologies, such as base editing [65][66][67][68][69] or prime editing 70 , both of which induce permanent modifications in the genome without double strand breaks, thus providing a significant advantage over Cas9 nuclease-based exon skipping techniques. We are hopefully that with the latest advances in technologies, powerful therapeutic approaches coupled with highly efficient delivery systems will be developed to achieve great potency, bioavailability and safety for USH2A disease, as well as other genetic diseases.…”

“…. Exon skipping can also be attained permanently by directly altering the genomic DNA, for instance using programmable CRISPR genome editing to delete the target exons or disrupt the specific splicing sites of the target exons37,38 .…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…Nuclease-induced DSBs are found to be detrimental to host cells by causing unwanted large fragment deletion [115] and p53-dependent DNA damage responses [116,117]. With the rapid development of novel applications [121,122] and new BE variants [123,124], the amount of targetable mutations is projected to expand dramatically. It is estimated that 60% of pathogenic point mutations can be reversed by current BEs [118].…”

“…Current BE platforms appear to exert significant off-target activity at the RNA level [119,120]. With the rapid development of novel applications [121,122] and new BE variants [123,124], the amount of targetable mutations is projected to expand dramatically.…”

Modelling asymmetric count ratios in CRISPR screens to decrease experiment size and improve phenotype detection

“…Subsequent DNA repair via non-homologous end joining eventually leads to frame-shift mutations and premature stop-codons, nonsense-mediated RNA decay and finally gene knockout. Alternatively, it is possible to introduce more subtle perturbations such as altered splicing patterns 13,14 or quantitative modulation of gene expression 15 . To this end, modified CRISPR associated enzymes which function as epigenetic modifiers [16][17][18] , transcriptional modulators [19][20][21] or single-base editors 22,23 are used.…”