Precise and programmable C:G to G:C base editing in genomic DNA

AbstractMany genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.

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Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins

Nature Communications ◽

10.1038/s41467-021-21559-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Liwei Chen ◽

Jung Eun Park ◽

Peter Paa ◽

Priscilla D. Rajakumar ◽

Hong-Ting Prekop ◽

...

Keyword(s):

Base Excision Repair ◽

Target Genes ◽

Therapeutic Potential ◽

Excision Repair ◽

Cytidine Deaminase ◽

Genetic Diseases ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Base Excision ◽

Nucleotide Resolution

AbstractMany genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

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Somatic mutations and single nucleotide polymorphisms of base excision repair genes involved in the repair of 8-hydroxyguanine in damaged DNA

Cancer Letters ◽

10.1016/s0304-3835(01)00435-9 ◽

2001 ◽

Vol 166 (1) ◽

pp. 65-69 ◽

Cited By ~ 25

Author(s):

Kazuya Shinmura ◽

Satoru Yamaguchi ◽

Takayuki Saitoh ◽

Takashi Kohno ◽

Jun Yokota

Keyword(s):

Single Nucleotide Polymorphisms ◽

Base Excision Repair ◽

Somatic Mutations ◽

Excision Repair ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Base Excision ◽

Repair Genes ◽

Damaged Dna

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Single Nucleotide Polymorphisms of DNA Base-excision Repair Genes (APE1, OGG1 and XRCC1) Associated with Breast Cancer Risk in a Chinese Population

Asian Pacific Journal of Cancer Prevention ◽

10.7314/apjcp.2014.15.3.1133 ◽

2014 ◽

Vol 15 (3) ◽

pp. 1133-1140 ◽

Cited By ~ 17

Author(s):

Hao Luo ◽

Zheng Li ◽

Yi Qing ◽

Shi-Heng Zhang ◽

Yu Peng ◽

...

Keyword(s):

Breast Cancer ◽

Base Excision Repair ◽

Chinese Population ◽

Excision Repair ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Dna Base Excision Repair ◽

Dna Base ◽

Base Excision ◽

Repair Genes

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ecRESCUE: a novel ecDHFR-regulated RESCUE system with reduced RNA off-targeting activity

Cell Communication and Signaling ◽

10.1186/s12964-021-00759-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Yihan Wang ◽

Guo Li ◽

Xiangyang Li ◽

Yuzhe Wang ◽

Xingxu Huang ◽

...

Keyword(s):

Genetic Diseases ◽

Transcriptional Level ◽

In Vitro Experiments ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Considerable Potential ◽

Base Editing ◽

High Incidence ◽

Rescue System

AbstractThe currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity.

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ecRESCUE: a novel ecDHFR-regulated RESCUE system with reduced RNA off-targeting activity

10.21203/rs.3.rs-459640/v1 ◽

2021 ◽

Author(s):

Jianen Gao ◽

Yihan Wang ◽

Guo Li ◽

Xiangyang Li ◽

Yuzhe Wang ◽

...

Keyword(s):

Genetic Diseases ◽

Transcriptional Level ◽

In Vitro Experiments ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Considerable Potential ◽

Base Editing ◽

High Incidence ◽

Rescue System

Abstract The currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity.

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Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Molecular Neurobiology ◽

10.1007/s12035-016-9971-6 ◽

2016 ◽

Vol 54 (6) ◽

pp. 4150-4159 ◽

Cited By ~ 16

Author(s):

Piotr Czarny ◽

Dominik Kwiatkowski ◽

Monika Toma ◽

Joanna Kubiak ◽

Agnieszka Sliwinska ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Nucleotide Polymorphisms ◽

Recurrent Depression ◽

Single Nucleotide ◽

Base Excision ◽

Repair Genes ◽

Depression Disorder

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Association Between Genetic Variants in the Base Excision Repair Pathway and Outcomes After Hematopoietic Cell Transplant.

Blood ◽

10.1182/blood.v114.22.870.870 ◽

2009 ◽

Vol 114 (22) ◽

pp. 870-870 ◽

Cited By ~ 1

Author(s):

Mukta Arora ◽

Bruce Lindgren ◽

Saonli Basu ◽

Sriharsha Nagaraj ◽

Myron Gross ◽

...

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Cell Transplant ◽

Nucleotide Polymorphisms ◽

Disease Relapse ◽

Hematopoietic Cell Transplant ◽

Single Nucleotide ◽

Deleterious Alleles ◽

Base Excision ◽

One Year

Abstract Abstract 870 Introduction: Despite recent advances in technology and supportive care, hematopoietic cell transplant (HCT) continues to be associated with high morbidity and mortality. Since the therapies used in HCT induce DNA damage that is repaired by the base excision repair (BER) pathway we hypothesized that single nucleotide polymorphisms (SNPs) in BER genes may influence HCT outcomes. Methods: We evaluated the association between tagSNPs and functionally important SNPs (n= 179) in the BER pathway with transplant related mortality at one year (TRM) and disease relapse in a cohort of 470 recipients of allogeneic HCT for hematologic malignancies at the University of Minnesota between 1998 and 2007 from a HLA-identical sibling donor, HLA-matched or mismatched unrelated donor (URD) or single umbilical cord graft. Results: After adjustment for age at transplant, donor type, race, and conditioning regimen, four SNPs in OGGI, LIG3 and MUTYH1 genes (rs159153, rs3135974, rs3219463, rs3219476) were associated with increased risk of TRM whereas two SNPs in TDG gene (rs167715, rs2374327) were associated with decreased risk of TRM at one year (p≤0.01). Patients with increasing numbers of deleterious alleles in the BER pathway showed an increased cumulative incidence of TRM at one year (14% for ≤ 1 deleterious allele vs. 51% for ≥ 4 deleterious alleles; p<0.001). One SNP, rs3135974, in LIG3 gene was associated with decreased risk of disease relapse (p<0.001) post HCT. Conclusions: Single nucleotide polymorphisms in the BER pathway are significantly associated with both TRM and disease relapse, thus supporting the hypothesis that genetic polymorphisms in the BER pathway are involved in the pathophysiology of TRM and disease relapse. If confirmed in independent cohorts, these SNPs may indicate patients who might benefit from altered or less DNA damaging conditioning regimens. Disclosures: No relevant conflicts of interest to declare.

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