Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.

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

Emerging Topics in Life Sciences ◽

10.1042/etls20170021 ◽

2017 ◽

Vol 1 (2) ◽

pp. 161-168 ◽

Cited By ~ 2

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.

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Base editing in Streptomyces with Cas9-deaminase fusions

10.1101/630137 ◽

2019 ◽

Cited By ~ 5

Author(s):

Zhiyu Zhong ◽

Junhong Guo ◽

Liang Deng ◽

Li Chen ◽

Jian Wang ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Genetic Manipulation ◽

Strand Break ◽

High Fidelity ◽

Nucleotide Substitutions ◽

Base Editing ◽

Dna Double Strand Break ◽

Repair Template ◽

Adenine Base

AbstractConventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

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Base editing and prime editing in laboratory animals

Laboratory Animals ◽

10.1177/0023677221993895 ◽

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.

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

Communications Biology ◽

10.1038/s42003-019-0745-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 15

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.

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Genome Editing in Cereals: Approaches, Applications and Challenges

International Journal of Molecular Sciences ◽

10.3390/ijms21114040 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4040 ◽

Cited By ~ 4

Author(s):

Waquar A. Ansari ◽

Sonali U. Chandanshive ◽

Vacha Bhatt ◽

Altafhusain B. Nadaf ◽

Sanskriti Vats ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Crop Improvement ◽

Whole Genome Sequence ◽

Cereal Crops ◽

Sequence Information ◽

World Population ◽

Base Editing ◽

Crop Varieties ◽

Current Scenario

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

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Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

10.1101/2020.01.06.890244 ◽

2020 ◽

Author(s):

Rabinowitz Roy ◽

Abadi Shiran ◽

Almog Shiri ◽

Offen Daniel

Keyword(s):

Genome Editing ◽

Point Mutations ◽

Nucleotide Variation ◽

Single Nucleotide Variation ◽

Computational Tool ◽

Single Nucleotide ◽

Base Editing ◽

A Genome ◽

Wide Range ◽

Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

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Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops

International Journal of Molecular Sciences ◽

10.3390/ijms21031024 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1024 ◽

Cited By ~ 7

Author(s):

Florian Veillet ◽

Laura Perrot ◽

Anouchka Guyon-Debast ◽

Marie-Paule Kermarrec ◽

Laura Chauvin ◽

...

Keyword(s):

Plant Breeding ◽

Genome Editing ◽

Gene Function ◽

Physcomitrella Patens ◽

Agronomic Traits ◽

Function Analysis ◽

Single Amino Acid ◽

Base Substitution ◽

Base Editing ◽

Gene Function Analysis

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

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

Nature Biotechnology ◽

10.1038/s41587-021-00933-4 ◽

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.

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Efficient and Accurate Single-Base Genome Editing in Corynebacterium Glutamicum by Target-Mismatched CRISPR/Cpf1

10.21203/rs.3.rs-29964/v1 ◽

2020 ◽

Author(s):

Hyun Ju Kim ◽

Se Young Oh ◽

Sang Jun Lee

Keyword(s):

Corynebacterium Glutamicum ◽

Genome Editing ◽

Negative Selection ◽

Stop Codon ◽

Single Point ◽

Point Mutations ◽

Live Cells ◽

Single Base ◽

Base Editing ◽

Target Dna

Abstract Background CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in C. glutamicum.Results This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in Corynebacterium glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5–95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were accurately edited.Conclusions The present results indicate that the target-mismatched Cpf1 negative selection method is not only efficient in C. glutamicum, but also an accurate single-base genome editing method.

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Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

10.1101/498402 ◽

2018 ◽

Author(s):

Aki Hayashi ◽

Katsunori Tanaka

Keyword(s):

Fission Yeast ◽

Genome Editing ◽

High Frequency ◽

Target Genes ◽

Point Mutations ◽

Inducible Promoter ◽

Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Guide Rna

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the microhomology mediated end joining (MMEJ)-based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the MMEJ-mediated genome editing and found that the MMEJ-mediated method provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing microhomology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

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