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.

Recent Advances in CRISPR/Cas9 Directed Base Editing

2021 ◽  
Vol 21 ◽  
Author(s):  
Nan Liu ◽  
Lifang Zhou ◽  
Junyan , Qu ◽  
Shaohua Yao
Keyword(s):  
Recent Progress ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Clinical Applications ◽  
Great Promise ◽  
Base Editing ◽  
Recent Advances ◽  
Dna Base ◽  
Genomic Editing

: Recently, CRISPR based techniques had significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled irreversible conversion of specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, high efficiency of those editing tools also provided great promise in clinical applications. In this review, we discuss recent progress and challenges of base editing tools.

scholarly journals ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Jingke Xie ◽  
Xingyun Huang ◽  
Xia Wang ◽  
Shixue Gou ◽  
Yanhui Liang ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Multiple Point ◽  
Nucleotide Polymorphisms ◽  
Spacer Length ◽  
Endogenous Gene ◽  
Base Editing ◽  
Fetal Fibroblasts

Abstract Background Many favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site. Results In this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system. Conclusion The newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

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 Adenine Base Editing in vivo with a Single Adeno-Associated Virus Vector

2021 ◽  
Author(s):  
Han Zhang ◽  
Nathan Bamidele ◽  
Pengpeng Liu ◽  
Ogooluwa Ojelabi ◽  
Xin D. Gao ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Cell Types ◽  
Vector System ◽  
Adeno Associated Virus ◽  
Base Editing ◽  
Guide Rna ◽  
Gene Therapies ◽  
Compact Size ◽  
Adenine Base

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (~5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared to the well-characterized Streptococcus pyogenes Cas9-containing ABEs, Nme2-ABE possesses a distinct PAM (N4CC) and editing window, exhibits fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we showed that in vivo delivery of Nme2-ABE and its guide RNA by a single-AAV vector can revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

scholarly journals Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis

2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Jindong Hao ◽  
Chengshun Li ◽  
Chao Lin ◽  
Yang Hao ◽  
Xianfeng Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Apoptosis ◽  
Epigenetic Modification ◽  
Point Mutations ◽  
Base Editing ◽  
Non Coding Rna ◽  
Induce Cell Apoptosis ◽  
The Relationship ◽  
Adenine Base

Abstract Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T–A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.

scholarly journals Computer simulations explain mutation-induced effects on the DNA editing by adenine base editors

2020 ◽  
Vol 6 (10) ◽  
pp. eaaz2309 ◽  
Author(s):  
Kartik L. Rallapalli ◽  
Alexis C. Komor ◽  
Francesco Paesani
Keyword(s):  
Conformational Changes ◽  
Single Mutation ◽  
Base Editing ◽  
Functional Roles ◽  
Dna Base ◽  
Dna Editing ◽  
Dynamics Simulations ◽  
Editing Enzyme ◽  
Adenine Base

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.

scholarly journals In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

2021 ◽  
Author(s):  
Tanja Rothgangl ◽  
Melissa K. Dennis ◽  
Paulo J. C. Lin ◽  
Rurika Oka ◽  
Dominik Witzigmann ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Point Mutations ◽  
Density Lipoprotein ◽  
Negative Regulator ◽  
Base Editing ◽  
Guide Rna ◽  
Humoral Immune ◽  
Cholesterol Levels ◽  
Adenine Base

AbstractMost known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

scholarly journals Base editing and prime editing in laboratory animals

2021 ◽  
pp. 002367722199389
Author(s):  
Federico Caso ◽  
Benjamin Davies
Keyword(s):  
Genome Editing ◽  
Laboratory Animal ◽  
Genome Engineering ◽  
Point Mutations ◽  
Animal Research ◽  
Ease Of Use ◽  
Laboratory Animals ◽  
Genomic Change ◽  
Base Editing ◽  
Adenine Base

Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple ‘search and replace’ editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.

scholarly journals Ex vivo therapeutic base and prime editing using chemically derived hepatic progenitors in a mouse model of tyrosinemia type 1

2020 ◽  
Author(s):  
Yohan Kim ◽  
Jihyeon Yu ◽  
Sung-Ah Hong ◽  
Jeongyun Eom ◽  
Kiseok Jang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Ex Vivo ◽  
Genetic Diseases ◽  
The Body ◽  
Target Cells ◽  
Dna Base ◽  
Cell Sources ◽  
Model Mouse ◽  
Fumarylacetoacetate Hydrolase ◽  
Hereditary Tyrosinemia

SummaryDNA base editors and prime editing technology capable of therapeutic base conversion enable ex vivo gene editing therapy for various genetic diseases. For such therapy, it is critical that the target cells survive well both outside the body and after transplantation. In this regard, chemically derived stem/progenitor cells are attracting attention as the most useful cell sources for clinical trials. Here, we generate chemically derived hepatic progenitors from the hereditary tyrosinemia type1 model mouse (HT1-mCdHs) and successfully correct the disease causing mutation using both adenosine base editors (ABEs) and prime editing tools. After transplantation into HT1 mice, ABE-corrected HT1-mCdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased the survival rate of HT1 model mice, suggesting a safe and effective ex vivo gene editing therapy.

scholarly journals Phage Peptides Mediate Precision Base Editing with Focused Targeting Window

2020 ◽  
Author(s):  
Kun Jia ◽  
Yan-ru Cui ◽  
Shisheng Huang ◽  
Peihong Yu ◽  
Zhengxing Lian ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Single Point ◽  
Disease Model ◽  
Point Mutations ◽  
Gene Correction ◽  
Nucleotide Substitutions ◽  
Base Editing ◽  
A Cell ◽  
Single Point Mutations ◽  
Adenine Base

AbstractCytidine base editors (CBE) are novel genome engineering tools that can generate C-to-T nucleotide substitutions without introducing double-stranded breaks (DSBs). Instead of generating single-point mutations, CBEs induce nucleotide substitutions at wobble positions within the 20-nucleotide target site. A variety of strategies have been developed to improve the targeting scope and window of CBEs. Among these strategies, molecular switches that can temporally control CBE activities represent compelling options. In this study, we investigated the feasibility of using a bacteriophage-derived peptide, referred to as G8PPD, as the off-switch of CBEs. We showed that G8PPD could be employed to control the activities of and improve the targeting window of A3A and BE3 CBEs and adenine base editor 7.10 (ABE7.10). Notably, in a cell-based disease model of Marfan syndrome, G8PPD facilitated A3A CBE-based gene correction with a more focused targeting window and improved the percentage of perfectly edited gene alleles from less than 4% to more than 38% of the whole population. Our study presents the first peptide off-switch that can improve the targeting scope of CBEs, thus highlighting the importance of the temporal control of BE activity for precision base editing.

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