scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

scholarly journals Base and Prime Editing Technologies for Blood Disorders

2021 ◽  
Vol 3 ◽  
Author(s):  
Panagiotis Antoniou ◽  
Annarita Miccio ◽  
Mégane Brusson
Keyword(s):  
Genome Editing ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Great Promise ◽  
Blood Disorders ◽  
Major Drawback ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Advantages And Disadvantages

Nuclease-based genome editing strategies hold great promise for the treatment of blood disorders. However, a major drawback of these approaches is the generation of potentially harmful double strand breaks (DSBs). Base editing is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. The scope of base editing tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations, such as many blood disorders and might be more effective than approaches based on homology-directed repair, which is moderately efficient in hematopoietic stem cells, the target cell population of many gene therapy approaches. In this review, we describe the development and evolution of the base editing system and its potential to correct blood disorders. We also discuss challenges of base editing approaches–including the delivery of base editors and the off-target events–and the advantages and disadvantages of base editing compared to classical genome editing strategies. Finally, we summarize the recent technologies that have further expanded the potential to correct genetic mutations, such as the novel base editing system allowing base transversions and the more versatile prime editing strategy.

Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also discussed.

scholarly journals Application of the Modified Cytosine Base-Editing in the Cultured Cells of Bama Minipig

2021 ◽  
Author(s):  
Jia-sheng Pan ◽  
Zi-sheng Lin ◽  
Jian-cong Wen ◽  
Jian-feng Guo ◽  
Xia-hui Wu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Base Pair ◽  
Double Strand Breaks ◽  
Fibroblast Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Base ◽  
Lean Meat ◽  
Base Editing ◽  
Target Effects

Abstract Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

scholarly journals Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

2021 ◽  
Vol 22 (12) ◽  
pp. 6457
Author(s):  
Ho Joung Lee ◽  
Hyun Ju Kim ◽  
Sang Jun Lee
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Target Recognition ◽  
Whole Genome ◽  
Microbial Genomes ◽  
Single Base ◽  
Base Editing ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.

scholarly journals Biology and Application of Genome Editing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-22-SCI-22
Author(s):  
Feng Zhang
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Large Scale ◽  
Gene Knockout ◽  
Gene Activation ◽  
Base Editing ◽  
Cellular Processes ◽  
Equity Ownership ◽  
Rna Knockdown

Precision genome editing, which can be used to alter specific DNA sequences, is a powerful tool for understanding the molecular circuitry underlying cellular processes. Over the past several years, we and others have harnessed microbial CRISPR-Cas systems for use as platforms for a range of genome manipulations, including single and multiplex gene knockout, gene activation, and large-scale screening applications. Recently, we discovered and characterized several novel CRISPR systems that target RNA, including the CRISPR-Cas13 family. We developed a toolbox for RNA modulation based on Cas13, including methods for highly specific RNA knockdown, transcript imaging, and precision base editing. During our initial characterization of Cas13, we observed that Cas13 also exhibits so-called non-specific "collateral" RNase activity in vitro, which we capitalized on to create SHERLOCK, a highly sensitive and specific CRISPR diagnostic platform. We are continuing to refine and extend CRISPR-based technologies as well as explore microbial diversity to find new enzymes and systems that can be adapted for use as molecular biology tools and novel therapeutics. Disclosures Zhang: Arbor Biotechnologies: Consultancy, Equity Ownership; Sherlock Biosciences: Consultancy, Equity Ownership; Pairwise Plants: Consultancy, Equity Ownership; Beam Therapeutics: Consultancy, Equity Ownership; Editas Medicine: Consultancy, Equity Ownership.

scholarly journals Programmable single and multiplex base-editing in Bombyx mori using RNA-guided cytidine deaminases

2018 ◽  
Author(s):  
Yufeng Li ◽  
Sanyuan Ma ◽  
Le Sun ◽  
Tong Zhang ◽  
Jiasong Chang ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Nonsense Mutations ◽  
Knock Out ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Endogenous Genes ◽  
Genome Scale

ABSTRACTStandard genome editing tools (ZFN, TALEN and CRISPR/Cas9) edited genome depending on DNA double strand breaks (DSBs). A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks were developed recently, which have changed this status and have been quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites being knocked out by BE3 with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base-editing in an invertebrate model.

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 AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1786 ◽  
Author(s):  
Mingming Liang ◽  
Tingting Sui ◽  
Zhiquan Liu ◽  
Mao Chen ◽  
Hongmei Liu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Nuclease Activity ◽  
Inhibitory Effect ◽  
Great Promise ◽  
Genetic Changes ◽  
Base Editing ◽  
Precise Control ◽  
Wide Range ◽  
First Time ◽  
Target Effects

The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly “shut off” nuclease activity of both CBE and ABE systems.

scholarly journals Recent advances in the CRISPR genome editing tool set

2019 ◽  
Vol 51 (11) ◽  
pp. 1-11 ◽  
Author(s):  
Su Bin Moon ◽  
Do Yon Kim ◽  
Jeong-Heon Ko ◽  
Yong-Sam Kim
Keyword(s):  
Genome Editing ◽  
Routine Practice ◽  
Base Editing ◽  
Large Size ◽  
Effector Genes ◽  
Associated Proteins ◽  
Potential Applications ◽  
Tool Set ◽  
Target Activity ◽  
Single Effector

Abstract Genome editing took a dramatic turn with the development of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system. The CRISPR-Cas system is functionally divided into classes 1 and 2 according to the composition of the effector genes. Class 2 consists of a single effector nuclease, and routine practice of genome editing has been achieved by the development of the Class 2 CRISPR-Cas system, which includes the type II, V, and VI CRISPR-Cas systems. Types II and V can be used for DNA editing, while type VI is employed for RNA editing. CRISPR techniques induce both qualitative and quantitative alterations in gene expression via the double-stranded breakage (DSB) repair pathway, base editing, transposase-dependent DNA integration, and gene regulation using the CRISPR-dCas or type VI CRISPR system. Despite significant technical improvements, technical challenges should be further addressed, including insufficient indel and HDR efficiency, off-target activity, the large size of Cas, PAM restrictions, and immune responses. If sophisticatedly refined, CRISPR technology will harness the process of DNA rewriting, which has potential applications in therapeutics, diagnostics, and biotechnology.

scholarly journals In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway

2019 ◽  
Vol 5 (4) ◽  
pp. eaav3335 ◽  
Author(s):  
Yuan Cai ◽  
Tianlin Cheng ◽  
Yichuan Yao ◽  
Xiao Li ◽  
Yuqian Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetic Diseases ◽  
Double Strand Breaks ◽  
Photoreceptor Degeneration ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Phenotypic Rescue ◽  
Rod And Cone Photoreceptors ◽  
Endonuclease Enzyme

Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)–based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.

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