Adenine base editing in an adult mouse model of tyrosinaemia

Song, Chun‐Qing; Jiang, Tingting; Richter, Michelle F.; Rhym, Luke H.; Koblan, Luke W.; Zafra, María Paz; Schatoff, Emma M.; Doman, Jordan L.; Cao, Yusong; Dow, Lukas E.; Zhu, Lihua Julie; Anderson, Daniel G.; Liu, David R.; Yin, Hao; Xue, Wen

doi:10.1038/s41551-019-0357-8

Cited by 157 publications

(143 citation statements)

References 33 publications

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“…The A‐to‐G editing efficiency of PCSK9 in the liver by dual‐AAV split‐ABE‐Rma573 is ≈6% (Figure 4d,e), which might meet the requirement for treating recessive genetic diseases. [ 18a,23 ] Significant reductions of PCSK9 protein level (≈50% reduction, 100 mg mL −1 in control mice, 50 mg mL −1 in split‐ABE‐Rma573 targeted mice) and lower VLDL/LDL (very low‐density lipoproteins/low‐density lipoproteins) level (≈0.2–≈0.15 ng mL −1 ) were observed through AI‐MAST strategy (Figure 4f,g), without long‐term liver damage in 6–8 weeks post‐injection (Figure 4h). Our data further highlighted that the intein based dual‐AAV split‐ABE system could induce A‐to‐G editing in vivo, representing an important tool for investigating gene function in adult animal and gene therapy in vivo.…”

Section: Resultsmentioning

confidence: 99%

Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

et al. 2020

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The adenosine base editor (ABE) is able to catalyze A•T to C•G conversion efficiently and precisely in vivo, representing a new method for gene therapy. Adeno associated virus (AAV) is a well‐studied vector for gene delivery in vivo. However, due to the limited loading capacity of AAV vector (≈4800 bp), it is difficult to package ABE (≈5400 bp) into a single AAV. To tackle this problem, ABE can be split into two smaller parts through intein‐mediated protein trans‐splicing. Here, 14 different split sites of nCas9 (Cas9 nickase) in combination with three different inteins (Mxe, Npu, and Rma) are screened through a GFP‐based reporter system to identify novel split‐ABEs. After infecting HEK293T and HeLa cells with dual AAVs, two split‐ABEs (split‐ABE‐Rma573 and split‐ABE‐Rma674) that can edit the target gene efficiently are identified. Furthermore, these dual‐AAV split‐ABEs can effectively disrupt the splicing acceptor of PCSK9 in mouse liver and the splicing donor of NR2E3 in mouse retina through AI‐MAST strategy. This study provides two new split‐ABEs to investigate gene function in vivo and in gene therapy, representing a new method to treat diseases by precisely repairing point mutations or inactivating genes through the AI‐MAST strategy.

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Section: Resultsmentioning

confidence: 99%

Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

et al. 2020

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“…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 .…”

mentioning

confidence: 99%

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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“…injection method induced specific genomic correction of the Fah splicing mutation (Yin et al 2014;Song et al 2019;Yin et al 2016). Another group reported that the application of a combination of viral and lipid nanoparticle-mediated systems of delivery of the SpCas9 mRNA, gRNA, and repair template DNA enables specific correction of the Fah splicing mutation in a Fah5981SB mouse model .…”

Section: Mice and Ratsmentioning

confidence: 99%

Genome editing methods in animal models

Lee

Yoon

Kim

2020

Animal Cells and Systems

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Genetically engineered animal models that reproduce human diseases are very important for the pathological study of various conditions. The development of the clustered regularly interspaced short palindromic repeats (CRISPR) system has enabled a faster and cheaper production of animal models compared with traditional gene-targeting methods using embryonic stem cells. Genome editing tools based on the CRISPR-Cas9 system are a breakthrough technology that allows the precise introduction of mutations at the target DNA sequences. In particular, this accelerated the creation of animal models, and greatly contributed to the research that utilized them. In this review, we introduce various strategies based on the CRISPR-Cas9 system for building animal models of human diseases and describe various in vivo delivery methods of CRISPR-Cas9 that are applied to disease models for therapeutic purposes. In addition, we summarize the currently available animal models of human diseases that were generated using the CRISPR-Cas9 system and discuss future directions. ARTICLE HISTORY

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Adenine base editing in an adult mouse model of tyrosinaemia

Cited by 157 publications

References 33 publications

Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

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

Genome editing methods in animal models

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