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Open accessJournal ArticleDOI: 10.1038/S41467-021-21559-9

Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins

02 Mar 2021-Nature Communications (Springer Science and Business Media LLC)-Vol. 12, Iss: 1, pp 1384-1384
Abstract: Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

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20 results found


Journal ArticleDOI: 10.1038/S41587-021-00938-Z
Luke W. Koblan1, Mandana Arbab2, Mandana Arbab3, Mandana Arbab1  +23 moreInstitutions (4)
Abstract: Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.

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9 Citations


Open accessJournal ArticleDOI: 10.3390/IJMS22116072
Katarzyna Horodecka1, Markus Düchler1Institutions (1)
Abstract: The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.

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Topics: CRISPR (60%), Cas9 (56.99%)

4 Citations


Open accessJournal ArticleDOI: 10.1038/S41434-021-00263-9
24 May 2021-Gene Therapy
Topics: Prime (order theory) (54%)

4 Citations


Open accessJournal ArticleDOI: 10.1016/J.TPLANTS.2021.06.015
Mahmudul Hassan1, Mahmudul Hassan2, Yingxiao Zhang3, Guoliang Yuan1  +6 moreInstitutions (3)
Abstract: CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.

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Topics: CRISPR (60%), Genome editing (57.99%)

3 Citations


Open accessJournal ArticleDOI: 10.1016/J.GGEDIT.2021.100005
24 Apr 2021-
Abstract: Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors use nucleobase modification chemistry to efficiently and precisely incorporate single nucleotide variants (SNVs) into the genome of living cells. Two classes of DNA base editors currently exist: deoxycytidine deamination-derived editors (CBEs, which facilitate C•G to T•A mutations) and deoxyadenosine deamination-derived base editors (ABEs, which facilitate A•T to G•C mutations). More recently, the development of mitochondrial base editors allowed the introduction of C•G to T•A mutations into mitochondrial DNA as well. Base editors show great potential as therapeutic agents and research tools, and extensive studies have been carried out to improve upon the original base editor constructs to aid researchers in a variety of disciplines. Despite their widespread use, there are few publications that focus on elucidating the biological pathways involved during the processing of base editor intermediates. Because base editors introduce unique types of DNA damage products (a U•G mismatch with a DNA backbone nick for CBEs, and an I•T mismatch with a DNA backbone nick for ABEs) to facilitate genome editing, a deep understanding of the DNA damage repair pathways that facilitate or impede base editing represents an important aspect for the further expansion and improvement of the technologies. Here, we first review canonical deoxyuridine, deoxyinosine, and single-stranded break repair. Then, we discuss how interactions among these different repair processes can lead to different base editing outcomes. Through this review, we hope to promote thoughtful discussions on the DNA repair mechanisms of base editing, as well as help researchers in the improvement of the current base editors and the development of new base editors.

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Topics: Genome editing (55%)

3 Citations


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35 results found


Open accessJournal ArticleDOI: 10.1101/GR.849004
01 Jun 2004-Genome Research
Abstract: WebLogo generates sequence logos, graphical representations of the patterns within a multiple sequence alignment. Sequence logos provide a richer and more precise description of sequence similarity than consensus sequences and can rapidly reveal significant features of the alignment otherwise difficult to perceive. Each logo consists of stacks of letters, one stack for each position in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. WebLogo has been enhanced recently with additional features and options, to provide a convenient and highly configurable sequence logo generator. A command line interface and the complete, open WebLogo source code are available for local installation and customization.

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Topics: Sequence logo (71%), Multiple sequence alignment (56%), Sequence (medicine) (54%) ... show more

9,230 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE17946
Alexis C. Komor1, Yongjoo Kim2, Yongjoo Kim1, Michael S. Packer1  +5 moreInstitutions (2)
19 May 2016-Nature
Abstract: Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

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Topics: Genome editing (56%), DNA repair (55%), Point mutation (55%) ... show more

2,245 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE24644
23 Nov 2017-Nature
Abstract: The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

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Topics: Base pair (57.99%), DNA (56.99%), Point mutation (56%) ... show more

1,475 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE13011
Samuel H. Sternberg1, Sy Redding2, Martin Jinek1, Eric C. Greene3  +1 moreInstitutions (4)
06 Mar 2014-Nature
Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

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Topics: Protospacer adjacent motif (67%), Cas9 (63%), Base pair (61%) ... show more

1,348 Citations


Open accessJournal ArticleDOI: 10.1038/S41586-019-1711-4
21 Oct 2019-Nature
Abstract: Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.

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Topics: Genome editing (59%), Guide RNA (53%), Cas9 (53%)

1,111 Citations


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