New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

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The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

Scientific Reports ◽

10.1038/s41598-021-87669-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Minkyung Choi ◽

Jae-Young Yun ◽

Jun-Hyuk Kim ◽

Jin-Soo Kim ◽

Sang-Tae Kim

Keyword(s):

Arabidopsis Thaliana ◽

Cytidine Deaminase ◽

Biological Research ◽

Loss Of Function ◽

Nucleotide Substitutions ◽

Viral Origin ◽

Nonsense Mutations ◽

Base Editing ◽

Low Efficiency ◽

Viable System

AbstractCRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

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The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

10.1101/628107 ◽

2019 ◽

Author(s):

Florian Veillet ◽

Laura Chauvin ◽

Marie-Paule Kermarrec ◽

François Sevestre ◽

Mathilde Merrer ◽

...

Keyword(s):

Solanum Tuberosum ◽

Genome Editing ◽

Genome Engineering ◽

Basic Research ◽

Loss Of Function ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Discrete Variation ◽

Tetraploid Potato ◽

Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

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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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Comprehensive analysis of prime editing outcomes in human embryonic stem cells

10.1101/2021.04.12.439533 ◽

2021 ◽

Author(s):

Omer Habib ◽

Gizem Habib ◽

Gue-ho Hwang ◽

Sangsu Bae

Keyword(s):

Stem Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Cytidine Deaminase ◽

Embryonic Stem ◽

Cell Types ◽

Great Promise ◽

Nucleotide Substitutions ◽

Base Editing

Prime editing is a versatile and precise genome editing technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This technique holds great promise for the analysis of gene function, disease modeling, and the correction of pathogenic mutations in clinically relevant cells such as human pluripotent stem cells (hPSCs). Here we comprehensively tested prime editing in hPSCs by generating a doxycycline-inducible prime editing platform. Prime editing successfully induced all types of nucleotide substitutions and small insertions and deletions, similar to observations in other human cell types. Moreover, we compared prime editing and base editing for correcting a disease-related mutation in induced pluripotent stem cells derived form a patient with α 1-antitrypsin (A1AT) deficiency. Finally, whole-genome sequencing showed that, unlike the cytidine deaminase domain of cytosine base editors, the reverse transcriptase domain of a prime editor does not lead to guide RNA-independent off-target mutations in the genome. Our results demonstrate that prime editing in hPSCs has great potential for complementing previously developed CRISPR genome editing tools.

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Multiple gene substitution by Target-AID base-editing technology in tomato

Scientific Reports ◽

10.1038/s41598-020-77379-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Johan Hunziker ◽

Keiji Nishida ◽

Akihiko Kondo ◽

Sanae Kishimoto ◽

Tohru Ariizumi ◽

...

Keyword(s):

Target Gene ◽

Cytidine Deaminase ◽

Nucleotide Substitutions ◽

Efficient Tool ◽

Multiple Gene ◽

Base Editing ◽

Carotenoid Accumulation ◽

Multiple Genes ◽

Single Target ◽

Activation Induced Cytidine Deaminase

AbstractThe use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T0 lines, we obtained 10 independent T0 lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.

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Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

10.20944/preprints202109.0362.v1 ◽

2021 ◽

Author(s):

Soo-Young Yum ◽

Goo Jang ◽

Okjae Koo

Keyword(s):

Genome Editing ◽

Cytidine Deaminase ◽

Chromosomal Rearrangements ◽

Dna Breaks ◽

Base Editing ◽

Multiple Loci ◽

Double Strand Dna Breaks ◽

Protospacer Adjacent Motif ◽

Motif Site ◽

Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽

10.1128/mbio.00342-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiwei Hu ◽

Yannan Wang ◽

Qian Liu ◽

Yan Qiu ◽

Zhiyu Zhong ◽

...

Keyword(s):

Genome Editing ◽

Dna Breaks ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

Guide Rnas ◽

Double Stranded Dna ◽

Target Sites ◽

Guide Sequence ◽

Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

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Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.605768 ◽

2020 ◽

Vol 2 ◽

Author(s):

Limin Hu ◽

Olalekan Amoo ◽

Qianqian Liu ◽

Shengli Cai ◽

Miaoshan Zhu ◽

...

Keyword(s):

Genome Engineering ◽

Molecular Breeding ◽

Target Genes ◽

Agronomic Traits ◽

Crop Improvement ◽

Brassica Napus L ◽

Editing Efficiency ◽

Base Editing ◽

Expected Gain ◽

Genome Wide

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

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

BMC Biology ◽

10.1186/s12915-020-00866-5 ◽

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.

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