scholarly journals Simplified adenine base editors improve adenine base editing efficiency in rice

2019 ◽  
Vol 18 (3) ◽  
pp. 770-778 ◽  
Author(s):  
Kai Hua ◽  
Xiaoping Tao ◽  
Weiyi Liang ◽  
Zhaoxia Zhang ◽  
Runyu Gou ◽  
...  
Keyword(s):  
Editing Efficiency ◽  
Base Editing ◽  
Adenine Base

scholarly journals Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Minh Thuan Nguyen Tran ◽  
Mohd Khairul Nizam Mohd Khalid ◽  
Qi Wang ◽  
Jacqueline K. R. Walker ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Dna ◽  
Dna Editing ◽  
Target Activity ◽  
Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

scholarly journals Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing

2021 ◽  
Author(s):  
Ha Rim Shin ◽  
Ji-Eun See ◽  
Jiyeon Kweon ◽  
Heon Seok Kim ◽  
Gi-Jun Sung ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Genome Engineering ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
Fluorescent Reporter ◽  
Editing Efficiency ◽  
Base Editing ◽  
Adenine Base

Abstract CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.

scholarly journals ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

2021 ◽  
Vol 22 (11) ◽  
pp. 5663
Author(s):  
Zupeng Wang ◽  
Xiaoying Liu ◽  
Xiaodong Xie ◽  
Lei Deng ◽  
Hao Zheng ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Nicotiana Benthamiana ◽  
Expression Cassette ◽  
Editing Event ◽  
Online Tool ◽  
Editing Efficiency ◽  
Base Editing ◽  
Single Transcript ◽  
Target Effect ◽  
Adenine Base

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.

scholarly journals Effective and precise adenine base editing in mouse zygotes

2018 ◽  
Vol 9 (9) ◽  
pp. 808-813 ◽  
Author(s):  
Puping Liang ◽  
Hongwei Sun ◽  
Xiya Zhang ◽  
Xiaowei Xie ◽  
Jinran Zhang ◽  
...  
Keyword(s):  
Base Editing ◽  
Mouse Zygotes ◽  
Adenine Base

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

2021 ◽  
Vol 4 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Pin Lyu ◽  
Zuyan Lu ◽  
Sung-Ik Cho ◽  
Manish Yadav ◽  
Kyung Whan Yoo ◽  
...  
Keyword(s):  
Base Editing ◽  
Adenine Base

scholarly journals Rescue of STAT3 Function in Hyper-IgE Syndrome Using Adenine Base Editing

2021 ◽  
Vol 4 (2) ◽  
pp. 178-190
Author(s):  
Andreas C. Eberherr ◽  
Andre Maaske ◽  
Christine Wolf ◽  
Florian Giesert ◽  
Riccardo Berutti ◽  
...  
Keyword(s):  
Base Editing ◽  
Hyper Ige Syndrome ◽  
Adenine Base

scholarly journals One-step multiple site-specific base editing by direct embryo injection for precision and pyramid pig breeding

2020 ◽  
Author(s):  
Ruigao Song ◽  
Yu Wang ◽  
Qiantao Zheng ◽  
Jing Yao ◽  
Chunwei Cao ◽  
...  
Keyword(s):  
Stop Codon ◽  
Point Mutations ◽  
Embryonic Cells ◽  
Site Specific ◽  
Editing Efficiency ◽  
Base Editing ◽  
One Step ◽  
Pig Performance ◽  
Novel Alleles

AbstractPrecise and simultaneous acquisition of multiple beneficial alleles in the genome to improve pig performance are pivotal for making elite breeders. Cytidine base editors (CBEs) have emerged as powerful tools for site-specific single nucleotide replacement. Here, we compare the editing efficiency of four CBEs in porcine embryonic cells and embryos to show that hA3A-BE3-Y130F and hA3A-eBE3-Y130F consistently results in higher base-editing efficiency and lower toxic effects to in vitro embryo development. We also show that zygote microinjection of hA3A-BE3-Y130F results in one-step generation of pigs (3BE pigs) harboring C-to-T point mutations, including a stop codon in CD163 and in MSTN and induce beneficial allele in IGF2. The 3BE pigs showed improved growth performance, hip circumference, food conversion rate. Our results demonstrate that CBEs can mediate high throughput genome editing by direct embryo microinjection. Our approach allows immediate introduction of novel alleles for beneficial traits in transgene-free animals for pyramid breeding.

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

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.

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 A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Bo Li ◽  
Naixia Ren ◽  
Lele Yang ◽  
Junhao Liu ◽  
Qilai Huang
Keyword(s):  
Single Cell ◽  
Genome Editing ◽  
Cell Clone ◽  
Editing Efficiency ◽  
Base Editing ◽  
Single Cell Clone ◽  
Cell Clones ◽  
Nucleotide Mismatch

AbstractCRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

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