CRISPR-Cas-Mediated Single Base Editing at More than One Locus in Rice Genome

2020 ◽  
pp. 51-62
Author(s):  
Kutubuddin A. Molla ◽  
Yinong Yang
Keyword(s):  
Rice Genome ◽  
Single Base ◽  
Base Editing

Single base editing of rs12603332 on Chromosome 17q21 with a Cytosine Base Editor regulates ORMDL3 and ATF6α expression

Allergy ◽  
2021 ◽  
Author(s):  
Ning Weng ◽  
Marina Miller ◽  
Alexa K. Pham ◽  
Alexis C. Komor ◽  
David H. Broide
Keyword(s):  
Single Base ◽  
Base Editing

scholarly journals Heterozygous IDH1R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

Oncogene ◽  
2018 ◽  
Vol 37 (38) ◽  
pp. 5160-5174 ◽  
Author(s):  
Shuang Wei ◽  
Jie Wang ◽  
Olutobi Oyinlade ◽  
Ding Ma ◽  
Shuyan Wang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Astroglial Cell ◽  
Single Base ◽  
Base Editing

scholarly journals A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
A. St. Martin ◽  
D. J. Salamango ◽  
A. A. Serebrenik ◽  
N. M. Shaban ◽  
W. L. Brown ◽  
...  
Keyword(s):  
Single Base ◽  
Base Editing

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 Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

2021 ◽  
Vol 22 (12) ◽  
pp. 6457
Author(s):  
Ho Joung Lee ◽  
Hyun Ju Kim ◽  
Sang Jun Lee
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Target Recognition ◽  
Whole Genome ◽  
Microbial Genomes ◽  
Single Base ◽  
Base Editing ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.

scholarly journals 216 Multiplex base editing of NK cell to enhance cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A229-A229
Author(s):  
Minjing Wang ◽  
Mitchell Kluesner ◽  
Patricia Claudio Vázquez ◽  
Beau Webber ◽  
Branden Moriarity
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
High Efficiency ◽  
Cell Function ◽  
Nk Cell ◽  
Functional Assay ◽  
Single Base ◽  
Editing Efficiency ◽  
Base Editing

BackgroundNatural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody-dependent cell-mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. NK cells are also known for their technical and biological challenges which result in low editing efficiencies, compared to T cells and other immune cells.MethodsBase editing (BE) is a CRISPR-Cas9 based genome editing technology that allows precise single base transitions. Previously, we reported a high efficiency method for multiplex engineering of T cells using BE and thus reasoned that applying similar concepts in NK cells may offer an opportunity to alter many genes simultaneously at higher efficiency through multiplex base editing. We thus selected a panel of genes bearing critical roles in NK cell function for immunotherapy, including inhibitory intracellular regulator AHR and CISH, inhibitory checkpoint receptor KLRG1, TIGIT, KLRC1, and PDCD1, and Fc receptor CD16A. CD16A is responsible for NK cell ADCC and is regulated via cleavage upon NK activation. Non-cleavable CD16A improves ADCC killing and can be achieved through single-base substitution with BE.ResultsUsing the adenosine BE (ABE8e), we achieved multiplex editing (6 genes) rates up to 99% and 95% editing/knockout at DNA and protein levels, respectively. Notably, we assessed for reduction in editing efficiency when additional genes were targeted and found no significant reduction in editing efficiencies when targeting up to 6 genes simultaneously. Moreover, functional evaluation of non-cleavable CD16A NK cells revealed up to 35% increase of cytotoxicity against Raji cells.ConclusionsWe were able to achieve high multiplex editing efficiency in primary human NK cells using ABE8eand there was no significant decrease of editing efficiency as the number of gene of interest increases, up to 6 genes in total. Functional assay confirmed increased NK cell cytotoxicity against tumor cells. Our end goal is to achieve high efficiency multiplex editing in CAR-expressing NK cells to further improve NK cell activity and toxicity for cancer immunotherapy.ReferenceWebber B, Lonetree C, Kluesner M, et al. Highly efficient multiplex human T cell engineering without double-strand breaks usingCas9 base editors. Nat Commun 2019;10:5222.

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

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.

scholarly journals Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

2021 ◽  
Author(s):  
Xiang-xing Zhu ◽  
Jia-sheng Pan ◽  
Tao Lin ◽  
Ye-cheng Yang ◽  
Qiu-yan Huang ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Gene Knockout ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Single Base ◽  
Base Editing ◽  
Adenine Base

Abstract Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

scholarly journals Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

2019 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Target Site ◽  
Gene Repression ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

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.

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