Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Christie, Kathleen A.; Courtney, David; DeDionisio, Larry; Chao-Shern, Connie; Majumdar, Shyamasree De; Mairs, Laura; Nesbit, M. Andrew; Moore, Tara

doi:10.1038/s41598-017-16279-4

Cited by 71 publications

(56 citation statements)

References 43 publications

(51 reference statements)

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“…This general concept of silencing the single disease-causing allele has been proven to be effective in a number of knockdown studies by antisense oligonucleotides (Seyhan 2011). Although the use of Cas9 to silence a gene has not been fully explored (Kolli et al 2017;Christie et al 2017;Bakondi et al 2016), it is theoretically possible to use Cas9-mediated knockout to replace antisense oligonucleotides for gene silencing.…”

Section: Diseases Benefitting From In Vivo Gene Editingmentioning

confidence: 99%

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

et al. 2019

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Progress in targeted gene editing by programmable endonucleases has paved the way for their use in gene therapy. Particularly, Cas9 is an endonuclease with high activity and flexibility, rendering it an attractive option for therapeutic applications in clinical settings. Many disease-causing mutations could potentially be corrected by this versatile new technology. In addition, recently developed switchable Cas9 variants, whose activity can be controlled by an external stimulus, provide an extra level of spatiotemporal control on gene editing and are particularly desirable for certain applications. Here, we discuss the considerations and difficulties for implementing Cas9 to in vivo gene therapy. We put particular emphasis on how switchable Cas9 variants may resolve some of these barriers and advance gene therapy in the clinical setting.

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Section: Diseases Benefitting From In Vivo Gene Editingmentioning

confidence: 99%

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

et al. 2019

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“…The CRISPR/Cas genome-editing system is highly specific, with the ability to discriminate between similar genomic sites, even alleles, based on a single nucleotide difference 1 . In order to target a genomic region with the CRISPR system, a single-guide RNA (sgRNA) must be designed that is specific to the region of interest.…”

Section: Main Textmentioning

confidence: 99%

“…A genetic variant can impact sgRNA sites by being located in or near a protospacer adjacent motif (PAM site), potentially generating or eliminating sgRNA sites in an individual. Accordingly, a heterozygous variant can generate an allele-specific sgRNA site by enabling differentiation of sgRNAs between alleles 11,12 (Figure 1a). Because Cas nucleases have different PAM sequences, a variant may create an allelespecific sgRNA site for one Cas but not another.…”

Section: Main Textmentioning

confidence: 99%

AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design

Keough

Lyalina

Olvera

et al. 2018

Preprint

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15The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing 16 by even a single base pair. However, current tools only design sgRNAs for a reference genome, not taking 17 into account individual variants which may generate, remove, or modify CRISPR/Cas sgRNA sites. This 18 may cause mismatches between designed sgRNAs and the individual genome they are intended to target, 19 leading to decreased experimental performance. Here we describe AlleleAnalyzer, a tool for designing 20 personalized and allele-specific sgRNAs for genome editing. We leverage >2,500 human genomes to 21 identify optimized pairs of sgRNAs that can be used for human therapeutic editing in large populations in 22 the future. 23 24 25 2 Keywords 26 27 CRISPR, sgRNA design, genomics, genome surgery, genome editing, computational biology 28 Background 29The CRISPR/Cas genome-editing system is highly specific, with the ability to discriminate between similar 30 genomic sites, even alleles, based on a single nucleotide difference [1]. In order to target a genomic region 31 with the CRISPR system, a single-guide RNA (sgRNA) must be designed that is specific to the region of 32 interest. While current sgRNA design tools incorporate various data relating to predicted efficiency and 33 specificity such as epigenetic marks and chromatin accessibility [2][3][4], in the vast majority of cases, sgRNAs 34 are designed using reference genomes, such as the hg38 assembly for human or the GRCm38 assembly for 35 mouse. Since sgRNAs are often used on cell lines or organisms with many nucleotide differences from the 36 reference (e.g., on average 0.1% of a human genome [5]). Despite the finding that sgRNAs can sometimes 37 tolerate a single basepair mismatch, these mismatches frequently negatively impact sgRNA efficiency and 38 render imprecise the results of specificity prediction [2, 6, 7]. Furthermore, the use of CRISPR to research 39 areas such as haploinsufficiency, genomic imprinting, and dominant negative diseases require allele-40 specific sgRNA design. To address these challenges, we developed AlleleAnalyzer, a software tool that 41 designs personalized and allele-specific sgRNAs for individual genomes, identifies pairs of sgRNAs to 42 generate excisions likely to block expression of a gene, and leverages patterns of shared variation from 43 >2,500 human genomes to design sgRNA pairs for that will have the greatest utility in a target population. 44 45 Results and Discussion 46 47 Incorporating genetic variation into sgRNA design enables personalized and allele-specific CRISPR All possible personalized sgRNAs for SpCas9, SaCas9 and cpf1 (Cas12a) in the region surrounding the 404 first exon of RHO WTC (Supplementary Figure 6). WTC has no homozygous variants in this region, thus 405 allele frequency and variant-related columns are blank. However, the sgRNAs are designed to avoid the 9 406 heterozygous variants that WTC has in this region. 407408 Supplementary Table 4

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“…So far, allele-specific CRISPR has been increasingly employed in treating various diseases such as retinitis pigmentosa [5][6][7][8], corneal dystrophy [9], dominant progressive hearing loss [10] and multiple cancers [11][12][13], as well as genome imprinting diseases [14]. And it was also used to alleviate haploinsufficiency by allele-specific CRISPR activation of wildtype alleles [15], and even was designed for manipulating human leukocyte antigen (HLA) locus [16].…”

Section: Introductionmentioning

confidence: 99%

“…However, it is always labor intensive and time consuming to figure out appropriate guide sequences that may discriminate between two alleles [9,17]. Currently, most web servers only design sgRNAs from the reference genomes, without allele discriminations.…”

Section: Introductionmentioning

confidence: 99%

AsCRISPR: a web server for allele-specific sgRNA design in precision medicine

Zhao

Tang

2019

Preprint

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Allele-specific targeting by CRISPR provides a point of entry for personalized gene therapy of dominantly inherited diseases, by selectively disrupting the mutant alleles or diseasecausing single nucleotide polymorphisms (SNPs), ideally while leaving normal alleles intact. Despite unprecedented specificity and tremendous therapeutic utility of allelespecific targeting by CRISPR, few bioinformatic tools have been implemented for the allele-specific purpose. We thus developed AsCRISPR (Allele-specific CRISPR), a web tool to aid the design of guide sequences that can discriminate between alleles. It can process with query sequences harboring single-base or short insertion-deletion (indel) mutations, as well as heterozygous SNPs deposited in the dbSNP database. Multiple CRISPR nucleases and their engineered variants including newly-developed Cas12b and CasX are included for users' choice. AsCRISPR provides the downloadable results of candidate guide sequences that may selectively target either allele. Meanwhile, AsCRISPR evaluates the on-target efficiencies, specificities and potential off-targets of those candidates, and also displays the restriction enzyme sites that might be disrupted upon successful genome edits. Other than designing allele-specific guide sequences for treating diseases, AsCRISPR could also be exploited to help studying the potential functions of genetic variants at certain gene loci by applying allele-specific editings. AsCRISPR is freely available at http://www.genemed.tech/ascrispr.

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Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Cited by 71 publications

References 43 publications

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications

AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design

AsCRISPR: a web server for allele-specific sgRNA design in precision medicine

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