scholarly journals Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions

2019 ◽  
Author(s):  
Zhiquan Liu ◽  
Siyu Chen ◽  
Huanhuan Shan ◽  
Mao Chen ◽  
Yuning Song ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Context Specificity ◽  
Gene Modification ◽  
Base Editing ◽  
Human Apobec3g ◽  
Activity Window

AbstractCytidine base editors, composed of a cytidine deaminase fused to Cas9 nickase, enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when more than one C is present in the activity window of cytidine deaminase, which negatively affects the precision. Here, we develop a new base editor with CC context-specificity using rationally engineered human APOBEC3G, thus significantly reduce unwanted bystander activities. In addition, efficient C-to-T conversion that can further recognize relaxed NG PAMs is achieved by combining an engineered SpCas9-NG variant. These novel base editors with improved precision and targeting scope will expand the toolset for precise gene modification in organisms.

scholarly journals Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Zhiquan Liu ◽  
Siyu Chen ◽  
Huanhuan Shan ◽  
Yingqi Jia ◽  
Mao Chen ◽  
...  
Keyword(s):  
Context Specificity ◽  
Base Editing ◽  
Human Apobec3g

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

2020 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
Jaiana Malabarba ◽  
Elisabeth Chevreau ◽  
Nicolas Dousset ◽  
Florian Veillet ◽  
Julie Moizan ◽  
...  
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Acetolactate Synthase ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Adventitious Regeneration ◽  
Base Editing ◽  
Perennial Plants ◽  
Guide Rnas ◽  
Albino Phenotype

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.

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

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.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

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.

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 A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

2018 ◽  
Author(s):  
Noah Jakimo ◽  
Pranam Chatterjee ◽  
Lisa Nip ◽  
Joseph M Jacobson
Keyword(s):  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Genome Engineering ◽  
Protein Domains ◽  
Human Cells ◽  
Gene Modification ◽  
Editing Efficiency ◽  
Base Editing ◽  
Protospacer Adjacent Motif

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased” nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

scholarly journals Stress tolerance enhancement via SPT15 base editing in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Yanfang Liu ◽  
Yuping Lin ◽  
Yufeng Guo ◽  
Fengli Wu ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Tolerance ◽  
Rna Polymerase Ii ◽  
Convex Surface ◽  
Cytidine Deaminase ◽  
Structure Alignment ◽  
Protein Structure Alignment ◽  
Base Editing ◽  
And Performance

Abstract Background Saccharomyces cerevisiae is widely used in traditional brewing and modern fermentation industries to produce biofuels, chemicals and other bioproducts, but challenged by various harsh industrial conditions, such as hyperosmotic, thermal and ethanol stresses. Thus, its stress tolerance enhancement has been attracting broad interests. Recently, CRISPR/Cas-based genome editing technology offers unprecedented tools to explore genetic modifications and performance improvement of S. cerevisiae. Results Here, we presented that the Target-AID (activation-induced cytidine deaminase) base editor of enabling C-to-T substitutions could be harnessed to generate in situ nucleotide changes on the S. cerevisiae genome, thereby introducing protein point mutation in cells. The general transcription factor gene SPT15 was targeted, and total 36 mutants with diversified stress tolerances were obtained. Among them, the 18 tolerant mutants against hyperosmotic, thermal and ethanol stresses showed more than 1.5-fold increases of fermentation capacities. These mutations were mainly enriched at the N-terminal region and the convex surface of the saddle-shaped structure of Spt15. Comparative transcriptome analysis of three most stress-tolerant (A140G, P169A and R238K) and two most stress-sensitive (S118L and L214V) mutants revealed common and distinctive impacted global transcription reprogramming and transcriptional regulatory hubs in response to stresses, and these five amino acid changes had different effects on the interactions of Spt15 with DNA and other proteins in the RNA Polymerase II transcription machinery according to protein structure alignment analysis. Conclusions Taken together, our results demonstrated that the Target-AID base editor provided a powerful tool for targeted in situ mutagenesis in S. cerevisiae and more potential targets of Spt15 residues for enhancing yeast stress tolerance.

scholarly journals CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary cells

2020 ◽  
Author(s):  
Mitchell G. Kluesner ◽  
Walker S. Lahr ◽  
Cara-Lin Lonetree ◽  
Branden A. Smeester ◽  
Patricia N. Claudio-Vázquez ◽  
...  
Keyword(s):  
Splice Site ◽  
Stop Codon ◽  
Cytidine Deaminase ◽  
Gene Knockout ◽  
Premature Stop Codon ◽  
Splice Sites ◽  
Immune Synapse ◽  
Base Editing ◽  
Human T Cells ◽  
Site Targeting

ABSTRACTBase editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we developed the program SpliceR (z.umn.edu/spliceR) to design cytidine-deaminase base editor (CBE) and adenosine-deaminase base editor (ABE) splice-site targeting guides. We compared the splice-site targeting and premature stop codon introduction in a knockout screen against the TCR-CD3 immune synapse in primary human T-cells. Our data suggests that 1) the CBE, BE4 is more reliable than the ABE, ABE7.10 for splice-site targeting knockout and 2) for both CBEs and ABEs, splice-donor targeting is the most reliable approach for base editing induced knockout.

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