Highly efficient RNA-guided base editing in mouse embryos

Kim, Kyoungmi; Ryu, Shi‐Yong; Kim, Sang‐Tae; Baek, Gayoung; Kim, Daesik; Lim, Kayeong; Chung, Eugene; Kim, Sung-Hyun; Kim, Jin-Soo

doi:10.1038/nbt.3816

Cited by 348 publications

(293 citation statements)

References 15 publications

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“…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 . 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 .…”

mentioning

confidence: 99%

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

Gaudelli

Komor

Rees

et al. 2017

Nature

3,090

2,886

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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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mentioning

confidence: 99%

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

Gaudelli

Komor

Rees

et al. 2017

Nature

3,090

2,886

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“…Recently, CRISPR/Cas9 machinery has been modified to induce a genetic change without introducing a doublestranded break, called Bbase editing.^This method utilizes cytidine deaminases and guide RNA to convert cytidine to uridine, resulting in a change to thymidine [15]. This technology has been used to efficiently introduce genetic changes into a variety of species, including plant, yeast, sea urchin, mouse zygotes, and human cells and tripronuclear zygotes [15][16][17][18][19][20][21][22][23][24][25][26]. While this technology still needs to be refined as mosaicism is still observed, it provides a new method that may introduce genetic changes more efficiently without having to damage DNA.…”

Section: Mybpc3mentioning

confidence: 99%

Editing the human genome: where ART and science intersect

Hershlag

Bristow

2018

J Assist Reprod Genet

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The rapid development of gene-editing technologies has led to an exponential rise in both basic and translational research initiatives studying molecular processes and investigating possible clinical applications. Early experiments using genome editing to study human embryo development have contradicted findings in studies on model organisms. Additionally, a series of four experiments over the past 2 years set out to investigate the possibilities of introducing genetic modifications to human embryos, each with varying levels of success. Here, we discuss the key findings of these studies, including the efficiency, the safety, the potential untoward effects, major flaws of the studies, and emerging alternative genome editing methods that may allow overcoming the hurdles encountered so far. Given these results, we also raise several questions about the clinical utilization of germline gene editing: For which indications is gene editing appropriate? How do gene-editing technologies compare with genetic testing methods currently used for screening embryos? What are the ethical considerations we should be concerned about? While further research is underway, and our understanding of how to implement this technology continues to evolve, it is critical to contemplate if and how it should be translated from the bench to clinical practice.

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“…Differing from the first generation of genome editing techniques, including ZFN (Zinc Finger Nucleases) and Talen (Transcription Activator-Like Effector Nucleases), the second generation of genome editing tool RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and more efficient, attracting more attentions [1,[5][6][7]. However, more efforts should be made to improve both the efficiency and the specificity of the RGEN [8].…”

Section: Introductionmentioning

confidence: 99%

Optimized Plasmid Construction Strategy for Cas9

Li²,

Hossen

et al. 2018

Cell Physiol Biochem

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Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.

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Highly efficient RNA-guided base editing in mouse embryos

Cited by 348 publications

References 15 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

Editing the human genome: where ART and science intersect

Optimized Plasmid Construction Strategy for Cas9

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