scholarly journals DNA capture by a CRISPR-Cas9–guided adenine base editor

Science ◽  
2020 ◽  
Vol 369 (6503) ◽  
pp. 566-571 ◽  
Author(s):  
Audrone Lapinaite ◽  
Gavin J. Knott ◽  
Cody M. Palumbo ◽  
Enrique Lin-Shiao ◽  
Michelle F. Richter ◽  
...  
Keyword(s):  
Structural Data ◽  
Bound State ◽  
Base Editing ◽  
Future Design ◽  
Dna Deamination ◽  
Dna Capture ◽  
Cellular Dna ◽  
Adenosine Deamination ◽  
Deaminase Domain ◽  
Adenine Base

CRISPR-Cas–guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo–electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA–like conformation. Furthermore, ABE8e’s accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

scholarly journals CRISPR-Cas9 DNA Base-Editing and Prime-Editing

2020 ◽  
Vol 21 (17) ◽  
pp. 6240 ◽  
Author(s):  
Ariel Kantor ◽  
Michelle McClements ◽  
Robert MacLaren
Keyword(s):  
Therapeutic Potential ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Target Cells ◽  
Therapeutic Success ◽  
Base Editing ◽  
Dna Base ◽  
Efficient Delivery ◽  
Cellular Dna ◽  
Adenine Base

Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.

scholarly journals Mapping CRISPR-Cas9 public and commercial innovation using The Lens institutional toolkit

2021 ◽  
Author(s):  
Osmat Azzam Jefferson ◽  
Simon Lang ◽  
Kenny Williams ◽  
Deniz Koellhofer ◽  
Aaron Ballagh ◽  
...  
Keyword(s):  
Public Investment ◽  
Open Data ◽  
Regulatory Elements ◽  
Policy Makers ◽  
Base Editing ◽  
The Public ◽  
Homology Directed Repair ◽  
A Genome ◽  
Institutional Capability ◽  
Cellular Dna

AbstractCRISPR-Cas9 is a revolutionary technology because it is precise, fast and easy to implement, cheap and components are readily accessible. This versatility means that the technology can deliver a timely end product and can be used by many stakeholders. In plant cells, the technology can be applied to knockout genes by using CRISPR–Cas nucleases that can alter coding gene regions or regulatory elements, alter precisely a genome by base editing to delete or regulate gene expression, edit precisely a genome by homology-directed repair mechanism (cellular DNA), or regulate transcriptional machinery by using dead Cas proteins to recruit regulators to the promoter region of a gene. All these applications can be for: 1) Research use (Non commercial), 2) Uses related product components for the technology itself (reagents, equipment, toolkits, vectors etc), and 3) Uses related to the development and sale of derived end products based on this technology. In this contribution, we present a prototype report that can engage the community in open, inclusive and collaborative innovation mapping. Using the open data at the Lens.org platform and other relevant sources, we tracked, analyzed, organized, and assembled contextual and bridged patent and scholarly knowledge about CRISPR-Cas9 and with the assistance of a new Lens institutional capability, The Lens Report Builder, currently in beta release, mapped the public and commercial innovation pathways of the technology. When scaled, this capability will also enable coordinated editing and curation by credentialed experts to inform policy makers, businesses and private or public investment.

scholarly journals Effective and precise adenine base editing in mouse zygotes

2018 ◽  
Vol 9 (9) ◽  
pp. 808-813 ◽  
Author(s):  
Puping Liang ◽  
Hongwei Sun ◽  
Xiya Zhang ◽  
Xiaowei Xie ◽  
Jinran Zhang ◽  
...  
Keyword(s):  
Base Editing ◽  
Mouse Zygotes ◽  
Adenine Base

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

2021 ◽  
Vol 4 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Pin Lyu ◽  
Zuyan Lu ◽  
Sung-Ik Cho ◽  
Manish Yadav ◽  
Kyung Whan Yoo ◽  
...  
Keyword(s):  
Base Editing ◽  
Adenine Base

scholarly journals BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

scholarly journals Rescue of STAT3 Function in Hyper-IgE Syndrome Using Adenine Base Editing

2021 ◽  
Vol 4 (2) ◽  
pp. 178-190
Author(s):  
Andreas C. Eberherr ◽  
Andre Maaske ◽  
Christine Wolf ◽  
Florian Giesert ◽  
Riccardo Berutti ◽  
...  
Keyword(s):  
Base Editing ◽  
Hyper Ige Syndrome ◽  
Adenine Base

scholarly journals Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Wide Range ◽  
Family Based ◽  
Correct Point ◽  
Adenine Base

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

scholarly journals Author Correction: Precision genome engineering through adenine base editing in plants

Nature Plants ◽  
2018 ◽  
Vol 4 (9) ◽  
pp. 730-730
Author(s):  
Beum-Chang Kang ◽  
Jae-Young Yun ◽  
Sang-Tae Kim ◽  
YouJin Shin ◽  
Jahee Ryu ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Base Editing ◽  
Adenine Base

scholarly journals Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shuo Li ◽  
Bo Yuan ◽  
Jixin Cao ◽  
Jingqi Chen ◽  
Jinlong Chen ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Base Editing ◽  
The Future ◽  
Docking Sites ◽  
Domain Insertion ◽  
Adenine Base

AbstractBase editing tools with diversified editing scopes and minimized RNA off-target activities are required for broad applications. Nevertheless, current Streptococcus pyogenes Cas9 (SpCas9)-based adenine base editors (ABEs) with minimized RNA off-target activities display constrained editing scopes with efficient editing activities at positions 4-8. Here, functional ABE variants with diversified editing scopes and reduced RNA off-target activities are identified using domain insertion profiling inside SpCas9 and with different combinations of TadA variants. Engineered ABE variants in this study display narrowed, expanded or shifted editing scopes with efficient editing activities across protospacer positions 2-16. And when combined with deaminase engineering, the RNA off-target activities of engineered ABE variants are further minimized. Thus, domain insertion profiling provides a framework to improve and expand ABE toolkits, and its combination with other strategies for ABE engineering deserves comprehensive explorations in the future.

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