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 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.

scholarly journals Developing PspCas13b-based enhanced RESCUE system, eRESCUE, with efficient RNA base editing

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Guo Li ◽  
Yihan Wang ◽  
Xiangyang Li ◽  
Yuzhe Wang ◽  
Xingxu Huang ◽  
...  
Keyword(s):  
Genetic Code ◽  
Rna Editing ◽  
Genetic Diseases ◽  
Disease Treatment ◽  
Editing Efficiency ◽  
Base Editing ◽  
Rescue System ◽  
Adenine Deaminase ◽  
Deaminase Domain

AbstractRNA base editing is potential for cellular function research and genetic diseases treating. There are two main RNA base editors, REPAIR and RESCUE, for in vitro use. REPAIR was developed by fusing inactivated Cas13 (dCas13) with the adenine deaminase domain of ADAR2, which efficiently performs adenosine-to-inosine (A-to-I) RNA editing. RESCUE, which performs both cytidine-to-uridine (C-to-U) and A-to-I RNA editing, was developed by fusing inactivated Cas13 (dCas13) with the evolved ADAR2. However, the relatively low editing efficiency of the RESCUE system limits its broad application. Here, we constructed an enhanced RESCUE (eRESCUE) system; this dPspCas13b-RESCUE-NES system was generated by fusing inactivated PspCas13b with the evolved ADAR2. We determined the endogenous mRNA A-to-I and C-to-U editing efficiency mediated by the dPspCas13b-RESCUE-NES system in HEK-293T cells. This new RNA base editor was then used to induce 177Ser/Gly conversion of inhibitor kappa B kinase β (IKKβ) by changing the genetic code from AGU to GGU. The results showed that the eRESCUE editor mediates more efficient A-to-I and C-to-U RNA editing than the RESCUE RNA editor, as was previously reported. The 177Ser/Gly conversion of IKKβ, accomplished by converting the genetic code from AGU to GGU, resulted in a decrease in the phosphorylation of IKKβ and downregulation of downstream IKKβ-related genes. In summary, we developed a more efficient RNA base editor, eRESCUE, which may provide a useful tool for biomedical research and genetic disease treatment.

scholarly journals Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

1990 ◽  
Vol 111 (6) ◽  
pp. 2537-2542 ◽  
Author(s):  
R Hinrichsen ◽  
E Wilson ◽  
T Lukas ◽  
T Craig ◽  
J Schultz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ion Channel ◽  
Cell Function ◽  
Point Mutations ◽  
Structural Features ◽  
Behavioral Phenotype ◽  
Amino Acid Side Chain ◽  
Chemical Homogeneity ◽  
Site Specific

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

scholarly journals One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

2021 ◽  
Vol 22 (5) ◽  
pp. 2249
Author(s):  
Fuminori Tanihara ◽  
Maki Hirata ◽  
Nhien Thi Nguyen ◽  
Osamu Sawamoto ◽  
Takeshi Kikuchi ◽  
...  
Keyword(s):  
Gene Editing ◽  
Multiple Gene ◽  
Knock Out ◽  
Editing Efficiency ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
One Step ◽  
Step Generation ◽  
The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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 In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x.

1996 ◽  
Vol 40 (1) ◽  
pp. 152-156 ◽  
Author(s):  
F Sifaoui ◽  
M D Kitzis ◽  
L Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
In Vitro Selection ◽  
Point Mutations ◽  
Fold Increase ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Beta Lactam Antibiotics ◽  
One Step ◽  
Resistant Mutants

Many oral penicillins and cephalosporins are used to treat clinical infections caused by Streptococcus pneumoniae. Therefore, using different beta-lactams as selectors, we estimated the frequencies of one-step mutations leading to resistance. Resistant mutants were obtained from penicillin-susceptible, intermediately resistant, and penicillin resistant strains. For cefixime, cefuroxime, cefpodoxime, cefotaxime, and ceftriaxone, the frequencies of mutation ranged from 10(-6) to 10(-8) when resistant mutants were selected at 2- to 8-fold the MIC, and the MICs increased 2- to 16-fold. For ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, cefaclor, and loracarbef, the frequencies of mutation were about 10(-7) to 10(-8), and the MICs increased twofold at most. One to three resistance profiles of the resulting mutants were selected for each of the selecting antibiotics. Among those, some showed resistance to the cephalosporins associated with a 2- to 32-fold increase in susceptibility to the penicillins. Competition experiments showed a decreased affinity of PBP2x for cefpodoxime in all mutants. In some mutants that were more susceptible to amoxicillin, a decreased affinity of PBP2x for cefpodoxime was associated with an increased affinity for amoxicillin and a particular substitution of alanine for threonine at position 550 just after the KSG triad. From these results we infer (i) that among the beta-lactams tested the penicillins, cefaclor, and loracarbef selected one-step resistant mutants less frequently and that they achieved a lower level of resistance, and (ii) that mutants with different profiles may have acquired different point mutations in PBP2x.

scholarly journals Functional Interaction of the Adenovirus IVa2 Protein with Adenovirus Type 5 Packaging Sequences

2005 ◽  
Vol 79 (5) ◽  
pp. 2831-2838 ◽  
Author(s):  
Philomena Ostapchuk ◽  
Jihong Yang ◽  
Ece Auffarth ◽  
Patrick Hearing
Keyword(s):  
Specific Interaction ◽  
Point Mutations ◽  
Adenovirus Type ◽  
Viral Proteins ◽  
Dna Packaging ◽  
Sequence Motif ◽  
Site Specific ◽  
Adenovirus Type 5

ABSTRACT Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.

scholarly journals Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

1998 ◽  
Vol 180 (20) ◽  
pp. 5437-5442 ◽  
Author(s):  
Annie Gravel ◽  
Nancy Messier ◽  
Paul H. Roy
Keyword(s):  
Dna Binding ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Wild Type ◽  
Conserved Residues ◽  
Site Specific ◽  
Gene Cassettes ◽  
In Vivo Recombination

ABSTRACT The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.

scholarly journals Sequence analysis and in vitro expression of genes 6 and 11 of an ovine group B rotavirus isolate, KB63: evidence for a non-defective, C-terminally truncated NSP1 and a phosphorylated NSP5

1999 ◽  
Vol 80 (8) ◽  
pp. 2077-2085 ◽  
Author(s):  
S. Shen ◽  
T. A. McKee ◽  
Z. D. Wang ◽  
U. Desselberger ◽  
D. X. Liu
Keyword(s):  
Stop Codon ◽  
Single Point ◽  
Point Mutations ◽  
Amino Acid Sequences ◽  
Nsp1 Gene ◽  
Functional Product ◽  
Genes Encoding ◽  
Group A Rotaviruses ◽  
Group B

An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People’s Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52·6–57·2%, the identities of deduced amino acid sequences were only 34·9–46·3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.

The Impact of Bioconjugation on the Interfacial Activity of a Protein Biosurfactant

2018 ◽  
Author(s):  
Hossam H Tayeb ◽  
Marina Stienecker ◽  
Anton Middelberg ◽  
Frank Sainsbury
Keyword(s):  
Polyethylene Glycol ◽  
Colloidal Stability ◽  
Point Mutations ◽  
Pharmaceutical Formulations ◽  
Industrial Processes ◽  
Parent Molecule ◽  
Site Specific ◽  
Interfacial Activity ◽  
Surface Active ◽  
The Impact

Biosurfactants, are surface active molecules that can be produced by renewable, industrially scalable biologic processes. DAMP4, a designer biosurfactant, enables the modification of interfaces via genetic or chemical fusion to functional moieties. However, bioconjugation of addressable amines introduces heterogeneity that limits the precision of functionalization as well as the resolution of interfacial characterization. Here we designed DAMP4 variants with cysteine point mutations to allow for site-specific bioconjugation. The DAMP4 variants were shown to retain the structural stability and interfacial activity characteristic of the parent molecule, while permitting efficient and specific conjugation of polyethylene glycol (PEG). PEGylation results in a considerable reduction on the interfacial activity of both single and double mutants. Comparison of conjugates with one or two conjugation sites shows that both the number of conjugates as well as the mass of conjugated material impacts the interfacial activity of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG conjugates to impart colloidal stability on peptide-stabilized emulsions is reduced. We suggest that this is due to constraints on the structure of amphiphilic helices at the interface. Specific and efficient bioconjugation permits the exploration and investigation of the interfacial properties of designer protein biosurfactants with molecular precision. Our findings should therefore inform the design and modification of biosurfactants for their increasing use in industrial processes, and nutritional and pharmaceutical formulations.

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