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.

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 BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

2020 ◽  
Author(s):  
András Tálas ◽  
Dorottya Simon ◽  
Péter Kulcsár ◽  
Éva Varga ◽  
Ervin Welker
Keyword(s):  
High Throughput ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Base ◽  
Base Editing ◽  
Target Effects

Abstract Adenine and cytosine base editors (ABE, CBE) are designed to generate single base mutations in genes without necessarily generating DNA double-strand breaks and undesired indel mutations. However, the activity of base editors employing an inactive (dead) SpCas9 is generally low, which may be increased only at the expense of generating undesired indels by using a nickase SpCas9. We have increased the efficiency of dead base editors to a level comparable to that of nickase base editors by enriching cells labelled for efficient base editing using Base Editor Activity Reporter (BEAR), a plasmid-based, fluorescent tool. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we have examined the applicability of increased fidelity variants to decrease Cas9-dependent off-target effects that revealed that CBE remains active on off-targets where increased fidelity mutations and/or mismatches decrease the activity of ABE, making the strategy of applying increased fidelity variants more beneficial for ABE than for CBE.

scholarly journals Depletion of Akt1 and Akt2 Impairs the Repair of Radiation-Induced DNA Double Strand Breaks via Homologous Recombination

2019 ◽  
Vol 20 (24) ◽  
pp. 6316 ◽  
Author(s):  
Tahereh Mohammadian Gol ◽  
H. Peter Rodemann ◽  
Klaus Dittmann
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Major Protein ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Strand Breaks ◽  
Protein Levels ◽  
Non Homologous End Joining

Homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and alternative NHEJ are major pathways that are utilized by cells for processing DNA double strand breaks (DNA-DSBs); their function plays an important role in the radiation resistance of tumor cells. Conflicting data exist regarding the role of Akt in homologous recombination (HR), i.e., the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT-knock-out and siRNA-mediated AKT-knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly demonstrated that HCT116 AKT1-KO and AKT2-KO cells have a significantly reduced Rad51 foci formation 6 h post irradiation versus parental cells. Depletion of Akt1 and Akt2 protein levels as well as inhibition of Akt kinase activity resulted in an increased number of residual-γH2AX in CENP-F positive cells mainly representing the S and G2 phase cells. Furthermore, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in stronger radiosensitivity of AKT1 and AKT2 knockout cells versus wild type cells. These data collectively show that both Akt1 and Akt2 are involved in DSBs repair through HRR.

scholarly journals CRISPR base editing applications for identifying cancer-driving mutations

2021 ◽  
Author(s):  
Martin Pal ◽  
Marco J. Herold
Keyword(s):  
Point Mutations ◽  
Double Strand Breaks ◽  
Great Promise ◽  
Functional Screening ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Dna Base ◽  
Damage Response ◽  
Dna Template

CRISPR base editing technology is a promising genome editing tool as (i) it does not require a DNA template to introduce mutations and (ii) it avoids creating DNA double-strand breaks, which can lead to unintended chromosomal alterations or elicit an unwanted DNA damage response. Given many cancers originate from point mutations in cancer-driving genes, the application of base editing for either modelling tumour development, therapeutic editing, or functional screening is of great promise. In this review, we summarise current DNA base editing technologies and will discuss recent advancements and existing hurdles for its usage in cancer research.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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