Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

Tang, Weixin; Hu, Johnny H.; Liu, David R.

doi:10.1038/ncomms15939

Cited by 195 publications

(140 citation statements)

References 37 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 . Fourth-generation base editors (BE4 and BE4-Gam) further improve editing efficiency and product purity 5 .…”

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

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3,220

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

Self Cite

3,220

2,886

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“…The Liu group embedded self-cleaving catalytic RNA aptazymes in the sgRNA, which block the sgRNA spacer sequence until exposure to a ligand (Tang et al, 2017). This makes base editing responsive to a ligand, although the system exhibited some activity without ligand and was somewhat less efficient than standard sgRNAs.…”

Section: Section 2: Applications Of Base Editingmentioning

confidence: 99%

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

et al. 2017

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The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double stranded breaks and donor templates), and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

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“…There are also numerous examples of gRNA that have been had additional functional RNA structures appended [(reviewed in (7)], including ribozymes at the 5′ end (12,14), modification of the upper stem with RNA aptamer binding effectors to colocalise fluorescent proteins (15), and 3′-fusion of viral RNA scaffolds to recruit a variety of transcriptional activators, suppressors, or protein modifiers for reprogramming gene expression (16). It is perhaps surprising given the sensitivity to even a single unpaired nucleotide that larger RNA structures can successfully accommodated at the 5′ end of Cas9 gRNAs [e.g.…”

Section: Introductionmentioning

confidence: 99%

5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Mullally

Aelst

Naqvi

et al. 2020

Preprint

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A key aim in exploiting CRISPR-Cas is the engineering of gRNA to introduce additional functionalities, ranging from small nucleotide changes that increase efficiency of on-target binding to the inclusion of large functional RNA aptamers and ribonucleoproteins (RNPs. Interactions between gRNA andCas9 are crucial for RNP complex assembly but several distinct regions of the gRNA are amenable to modification. Using a library of modified gRNAs, we used in vitro ensemble and single-molecule assays to assess the impact of RNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that R-loop formation and DNA cleavage activity are essentially unaffected by gRNA modifications of the Upper Stem, first Hairpin and 3′ end. In contrast, 5′ additions of only two or three nucleotides reduced R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such gRNA modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end supported formation of a stable ~9 bp R-loop that could not activate DNA cleavage. These observations will assist in successful gRNA design.

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Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

Cited by 195 publications

References 37 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

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

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