Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors

Huang, Tony P.; Zhao, Kevin T.; Miller, Shannon M.; Gaudelli, Nicole M.; Oakes, Benjamin L.; Fellmann, Christof; Savage, David F.; Liu, David R.

doi:10.1038/s41587-019-0134-y

Cited by 211 publications

(164 citation statements)

References 38 publications

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“…Although ABE variants recognizing non-NGG PAMs have been described [12][13][14] , editing efficiencies of these constructs are decreased in many instances when compared to outcomes observed with S. pyogenes Cas9 targeting NGG PAM sequences [12][13][14] . To determine whether our eighth-generation evolved deaminase also increases the editing efficiencies at target sites bearing non-NGG PAMs, we created ABE8 editors that replace S. pyogenes Cas9 with an engineered S. py.…”

Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

“…To date, seventh generation ABEs (ABE7) have enabled efficient A•T to G•C conversion in the genomes of humans 5 , mice [6][7][8] , bacteria 1 , plants 9,10 , and a variety of other species, reviewed here 11 . Many therapeutic targets, however, may benefit from a more active ABE with a broader editing window or improved compatibility with non-NGG nCas9s [12][13][14] as well as increased editing efficiencies in human cell lines 15 or when used in vivo 8 . The need for a more active version of ABE7.10 is the greatest when target adenines are positioned on the outer edges of the canonical ABE editing window (positions 3, 4, 7 and 8).…”

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confidence: 99%

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Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

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The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ∼1.5x higher editing at protospacer positions A5-A7 and ∼3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ∼4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

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Section: Abe8s With Either Non-ngg Pam Ncas9 Variants or Catalyticallmentioning

confidence: 99%

mentioning

confidence: 99%

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli

Lam

Rees

et al. 2020

Preprint

Self Cite

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“…However, the base editing field is rapidly evolving to expand the range of targetable codons. Recently, base editors encoding alternative Cas9 orthologs or engineered SpCas9 variants that recognize a broader range of PAMs have been optimized (Hu et al , ; Nishimasu et al , ; Huang et al , ; Kleinstiver et al , ). In parallel, CBEs have been developed with reduced or expanded width of the editing window, to minimize bystander editing at non‐target cytosines or to enlarge the repertoire of targetable bases, respectively (Kim et al , ; Jiang et al , ; Zafra et al , ; Tan et al , ; Thuronyi et al , ).…”

Section: Discussionmentioning

confidence: 99%

In situ CRISPR‐Cas9 base editing for the development of genetically engineered mouse models of breast cancer

et al. 2020

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Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR‐based gene disruption offers a fast‐track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock‐in mice with Cre‐conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA‐encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple‐negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.

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“…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].…”

Section: Single Nucleotide Mutations By Base Editorsmentioning

confidence: 99%

“…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.…”

Section: Single Nucleotide Mutations By Base Editorsmentioning

confidence: 99%

Adenovirus vectors in hematopoietic stem cell genome editing

Lieber

2019

FEBS Letters

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Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34 + cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.

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Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors

Cited by 211 publications

References 38 publications

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

In situ CRISPR‐Cas9 base editing for the development of genetically engineered mouse models of breast cancer

Adenovirus vectors in hematopoietic stem cell genome editing

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