Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity

Komor, Alexis C.; Zhao, Kevin T.; Packer, Michael S.; Gaudelli, Nicole M.; Waterbury, Amanda L.; Koblan, Luke W.; Kim, Yongjoo B.; Badran, Ahmed H.; Liu, David R.

doi:10.1126/sciadv.aao4774

Cited by 668 publications

(671 citation statements)

References 29 publications

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“…Taken together, these observations suggest that cellular repair of inosine intermediates created by ABEs is inefficient, obviating the need to subvert base excision repair. This situation contrasts with that of BE3 and BE4, which are strongly dependent on inhibiting uracil excision to maximize base editing efficiency and product purity 3,5 .…”

Section: Resultsmentioning

confidence: 91%

“…Our previously described base editors 3,5,7,8 exploit the use of cytidine deaminase enzymes that operate on single-stranded DNA but reject double-stranded DNA. This feature is critical to restrict deaminase activity to a small window of nucleotides within the single-stranded bubble created by Cas9.…”

Section: Resultsmentioning

confidence: 99%

“…Base editing is a form of genome editing that enables direct, irreversible conversion of one base pair to another at a target genomic locus without requiring double-stranded DNA breaks (DSBs), homology-directed repair (HDR) processes, or donor DNA templates 3–5 . Compared with standard genome editing methods to introduce point mutations, base editing can proceed more efficiently 3 , and with far fewer undesired products such as stochastic insertions or deletions (indels) or translocations 3–8 .…”

mentioning

confidence: 99%

“…The most commonly used base editors are third-generation designs (BE3) comprising ( i ) a catalytically impaired CRISPR-Cas9 mutant that cannot make DSBs, ( ii ) a single-strand-specific cytidine deaminase that converts C to uracil (U) within a ~5-nucleotide window in the single-stranded DNA bubble created by Cas9, ( iii ) a uracil glycosylase inhibitor (UGI) that impedes uracil excision and downstream processes that decrease base editing efficiency and product purity 5 , and ( iv ) nickase activity to nick the non-edited DNA strand, directing cellular DNA repair processes to replace the G-containing DNA strand 3,5 . Together, these components enable efficient and permanent C•G to T•A base pair conversion in bacteria, yeast 4,9 , plants 10,11 , zebrafish 8,12 , mammalian cells 3–8,13,14 , mice 8,15,16 , and even human embryos 17,18 .…”

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confidence: 99%

“…Base editing capabilities have expanded through the development of base editors with different protospacer-adjacent motif (PAM) compatibilities 7 , narrowed editing windows 7 , enhanced DNA specificity 8 , and small-molecule dependence 19 . Fourth-generation base editors (BE4 and BE4-Gam) further improve editing efficiency and product purity 5 .…”

mentioning

confidence: 99%

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Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Gaudelli

Komor

Rees

et al. 2017

Nature

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Summary The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C therefore could advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA. We evolved a tRNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs (e.g., ABE7.10), that convert target A•T to G•C base pairs efficiently (~50% in human cells) with very high product purity (typically ≥ 99.9%) and very low rates of indels (typically ≤ 0.1%). ABEs introduce point mutations more efficiently and cleanly than a current Cas9 nuclease-based method, induce less off-target genome modification than Cas9, and can install disease-correcting or disease-suppressing mutations in human cells. Together with our previous base editors, ABEs advance genome editing by enabling the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Gaudelli

Komor

Rees

et al. 2017

Nature

Self Cite

3,220

2,886

View full text Add to dashboard Cite

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Analysis of Pathogenic Variants Correctable With CRISPR Base Editing Among Patients With Recessive Inherited Retinal Degeneration

2021

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IMPORTANCE Many common inherited retinal diseases are not easily treated with gene therapy. Gene editing with base editors may allow the targeted repair of single-nucleotide transition variants in DNA and RNA. It is unknown how many patients have pathogenic variants that are correctable with a base editing strategy.OBJECTIVE To assess the prevalence and spectrum of pathogenic single-nucleotide variants amenable to base editing in common large recessively inherited genes that are associated with inherited retinal degeneration. DESIGN, SETTING, AND PARTICIPANTSIn this retrospective cross-sectional study, nonidentifiable records of patients with biallelic pathogenic variants of genes associated with inherited retinal degeneration between July 2013 and December 2019 were analyzed using data from the Oxford University Hospitals Medical Genetics Laboratories, the Leiden Open Variation Database, and previously published studies. Six candidate genes (ABCA4, CDH23, CEP290, EYS, MYO7A, and USH2A), which were determined to be the most common recessive genes with coding sequences not deliverable in a single adeno-associated viral vector, were examined. Data were analyzed from April 16 to May 11, 2020.MAIN OUTCOMES AND MEASURES Proportion of alleles with a pathogenic transition variant that is potentially correctable with a base editing strategy and proportion of patients with a base-editable allele.RESULTS A total of 12 369 alleles from the Leiden Open Variation Database and 179 patients who received diagnoses through the genetic service of the Oxford University Hospitals Medical Genetics Laboratories were analyzed. Editable variants accounted for 53% of all pathogenic variants in the candidate genes contained in the Leiden Open Variation Database. The proportion of pathogenic alleles that were editable varied by gene; 63.1% of alleles in ABCA4, 62.7% of alleles in CDH23, 53.8% of alleles in MYO7A, 41.6% of alleles in CEP290, 37.3% of alleles in USH2A, and 22.2% of alleles in EYS were editable. The 5 most common editable pathogenic variants of each gene accounted for a mean (SD) of 19.1% (9.5%) of all pathogenic alleles within each gene. In the Oxford cohort, 136 of 179 patients (76.0%) had at least 1 editable allele. A total of 53 of 107 patients (49.5%) with biallelic pathogenic variants in the gene ABCA4 and 16 of 56 patients (28.6%) with biallelic pathogenic variants in the gene USH2A had 1 of the 5 most common editable alleles.CONCLUSIONS AND RELEVANCE This study found that pathogenic variants amenable to base editing commonly occur in inherited retinal degeneration. These findings, if generalized to other cohorts, provide an approach for developing base editing therapies to treat retinal degeneration not amenable to gene therapy.

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Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR‐based screens

Wilson

Loizou

2022

Molecular Oncology

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The concepts of synthetic lethality and viability have emerged as powerful approaches to identify vulnerabilities and resistances within the DNA damage response for the treatment of cancer. Historically, interactions between two genes have had a longstanding presence in genetics and have been identified through forward genetic screens that rely on the molecular basis of the characterized phenotypes, typically caused by mutations in single genes. While such complex genetic interactions between genes have been studied extensively in model organisms, they have only recently been prioritized as therapeutic strategies due to technological advancements in genetic screens. Here, we discuss synthetic lethal and viable interactions within the DNA damage response and present how CRISPR-based genetic screens and chemical compounds have allowed for the systematic identification and targeting of such interactions for the treatment of cancer.

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Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity

Abstract: Probing base editing outcomes leads to new C:G to T:A base editors with greater efficiency and product purity, and fewer indels.

Cited by 668 publications

References 29 publications

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Analysis of Pathogenic Variants Correctable With CRISPR Base Editing Among Patients With Recessive Inherited Retinal Degeneration

Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR‐based screens

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