Respondida: Como verificar a edição genômica com tecnologia CRISPR/Cas9?

Fri, 19 Feb 2021 12:50:10 +0000

Pode ser efetuada a identificação de erros de pareamento com uso da enzima T7 endonuclease I (T7EI mismatch assay). Seguem informações relevantes sobre esse experimento, retiradas de HorizonDiscovery.com.

Considerations for T7 Endonuclease I (T7EI) mismatch assays
An overview of design rules, benefits, and limitations of T7EI detection for CRISPR experiments
T7 endonuclease I (T7EI) mismatch detection assays1 are commonly employed to evaluate the gene editing efficiency of CRISPR-Cas9 reagents at a given guide RNA target site in a population of edited cells. Here, we will provide an overview of the method, its advantages and limitations, and describe design considerations for T7EI primers.
Method overview
T7 endonuclease I recognizes and cleaves structural deformities in DNA heteroduplexes. In the context of a gene editing experiment, genomic DNA of a population of cells treated with CRISPR-Cas9 reagents will be amplified by PCR surrounding the CRISPR guide RNA target site. If a non-homologous end joining (NHEJ) repair event following CRISPR-Cas9 cleavage has introduced a mutation, denaturing and annealing will form a fraction of heteroduplexes of mutant and wildtype PCR amplicons. T7 endonuclease I will recognize and cleave DNA mismatches in those heteroduplexes. Running the cleavage products on a polyacrylamide or agarose gel, will resolve full length and cleavage products. The intensity of the respective bands will allow to calculate the gene editing percentage that has occurred (Figure 1).
Figure 1. Schematic and example of a DNA mismatch detection assay. Genomic DNA (blue) from edited cells contains wild type and edited DNA (mutation in red). PCR amplification around the editing site generates wild type and edited PCR products (black). Denaturing and reannealing of these products generates mismatches that are cleaved by the T7EI nuclease. Running these PCR products on a gel resolves full length DNA and cleavage products. Gel shows untreated (-) and cells edited with Cas9 and crRNA:tracrRNA (+).
Common rules for designing T7 mismatch primers for your gene of interest
PCR primers should surround the desired crRNA cleavage site. We suggest an amplicon size of about 500-800 nucleotides. To be easily resolvable by gel electrophoresis, the cleavage products should be of different sizes (e.g. 150base pairs and 350base pairs), with the CRISPR target site off-center.
Primer Tm should match the DNA polymerase that is used for amplification from gDNA. An example T7EI DNA mismatch detection assay protocol, including thermal cycling and cleavage conditions, utilizing Edit-R™ crRNA Control primer set and gDNA from direct cell lysis can be found here: T7EI mismatch detection assay protocol utilizing gDNA from direct cell lysis.
Utility and limitations
T7EI mismatch detection assays are a quick and cost-efficient means for estimating relative gene editing efficiency in a cell population, or for screening various clones and conditions. They are commonly used to optimize gene editing efficiencies in conjunction with validated guide RNA controls. While they can only assess the relative percentage of insertions and deletions (not functional protein knockout), they are highly useful for:
Optimizing Cas9/gRNA transfection conditions with a positive control or verifying your experimental conditions for good CRISPR-Cas9 gene editing in ongoing experiments.
Determining the optimal promoter for driving plasmid-based Cas9 Nuclease expression in your cell line of choice (Figure 2).
Choosing the most effective guide RNA for knock in experiments by homology-directed repair (HDR), which requires a high cleavage efficiency without the need for a functional knockout.
Differential expression of Cas9 by different promoters induce varying levels of gene editing
Figure 2. A human recombinant U2OS ubiquitin-EGFP proteasome cell line (Ubi[G76V]-EGFP) (A) and a mouse fibroblast (NIH/3T3) (B), were stably transduced with lentiviral particles containing Cas9 and a blasticidin resistance gene driven by the indicated promoters.. A population of cells with stably integrated Cas9-blastR was selected with blasticidin for a minimum of 10 days before transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Human PPIB / mouse Ppib using DharmaFECT 1 and DharmaFECT 3 Transfection reagent, respectively. After 72 hours, the relative frequency of gene editing was calculated based on a DNA mismatch detection assay using T7EI on genomic DNA extracted from the transfected cells.
To assess gene editing by T7EI, keep in mind that DNA cleavage and gene editing are not the same (See featured article: Proper assessment of gene editing with DNA mismatch detection assays). Also, mismatch detection nucleases are not 100% efficient at detecting all types of mismatches or bulges/distortions in DNA2. Likewise, large mutations that mutate the primer binding sites will not be detectable by T7EI mismatch assays.
When using DNA mismatch detection assay to screen mutated clonal lines, T7EI will not be able to recognize homoduplexes of mutant alleles. Clonal lines with the same mutation in all alleles will not be detected as these PCR products can reanneal and not be digested by the endonuclease (See application note: A CRISPR-Cas9 gene engineering workflow: generating functional knockouts using Dharmacon™ Edit-R™ Cas9 and synthetic crRNA and tracrRNA).
Further, the nature of the editing event will not be determined. If you require information on the nucleotide level, sequencing of clonal cell lines is the best method. Sequencing and further functional assays are recommended to fully characterize the impact of the editing event on gene function or cellular phenotype in your edited cell line.
References
R. D. Mashal, J. Koontz, J. Sklar, Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 9, 177-183 (1995).
L. Vouillot, A. Thelie, N. Pollet, Comparison of T7EI and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5, 407-415 (2015).
Author: Kathrin Kerschgens Ph.D. | Project Manager Cell Line Engineering
Additional Resources
CRISPR controls and detection primers
Ensure your system is optimized for successful CRISPR-Cas9 experiments
The mathematics behind DNA mismatch detection assays
Learn how to derive the equations behind mismatch detection assays
Beta tool: T7EI calculator
Automate your T7EI gel analysis to measure percent gene editing using our T7EI calculator!
Proper assessment of gene editing with DNA mismatch detection assays
This article shows why DNA cleavage and gene editing are not the same and highlights the importance of properly calculating gene editing percentages.
Microinjection of zebrafish embryos using Dharmacon™ Edit-R™ Cas9 Nuclease mRNA, synthetic crRNA, and tracrRNA for genome engineering
Application example for a T7EI mismatch assay

Quais as propriedades da ZsGreen1 GFP?

Thu, 09 Mar 2017 20:18:15 +0000

Informações do site da Takara Clontech (vide https://goo.gl/auFJkZ):

ZsGreen1 Fluorescent Protein
Living Colors ZsGreen1 is a bright green fluorescent protein derived from a Zoanthus sp. reef coral (1) that has been modified for high solubility, bright emission, and rapid chromophore maturation. The ZsGreen1 fluorescent protein has been reported to have better expression than EGFP in lung and muscle tissue and in cultured cells (2).
Features
Exceptional brightness: 2.5 times brighter than EGFP
ZsGreen1 excitation and emission maxima are 493 and 505 nm, respectively
References
Matz, M. V. et al. (1999) Nature Biotechnol. 17(10):969–973. Erratum in: Nature Biotechnol. (1999) 17(12):1227. Fluorescent proteins from nonbioluminescent Anthozoa species.
Bell, P. et al. (2007) J. Histochem. Cytochem. 55(9):931–939. A comparative analysis of novel fluorescent proteins as reporters for gene transfer studies.

Respondida: Quais as principais estratégias para manipulação gênica e clonagem molecular?

Fri, 17 Feb 2017 22:26:05 +0000

As estratégias mais comuns explicadas em uma série de artigos da IDT DNA:

Cloning strategies, Part 1: Assembly PCR for novel gene synthesis - Learn how you can use single-stranded oligos or a mix of single- and double-stranded DNA to produce longer genes of up to several thousand base pairs. No restriction sites are needed, and the approach is beneficial for assembling constructs that contain modular elements, such as antibodies.

Cloning strategies, Part 2: Cohesive-end cloning - Cohesive-end cloning is one of the most commonly employed techniques in molecular biology. Review these tips and tricks for cloning using restriction enzymes.

Cloning strategies, Part 3: Blunt-end cloning - Cloning of double-stranded DNA into plasmid vectors is one of the most commonly employed techniques in molecular biology. The procedure is used for sequencing, building libraries of DNA molecules, expressing coding and non-coding RNA, and many other applications. Learn about blunt-end cloning as a third method by which DNA fragments can be cloned into a plasmid vector.

IEC - Perguntas e respostas recentes em Manipulação genética

Respondida: Como verificar a edição genômica com tecnologia CRISPR/Cas9?

Considerations for T7 Endonuclease I (T7EI) mismatch assays

Method overview

Common rules for designing T7 mismatch primers for your gene of interest

Utility and limitations

Differential expression of Cas9 by different promoters induce varying levels of gene editing

References

Additional Resources

CRISPR controls and detection primers

The mathematics behind DNA mismatch detection assays

Beta tool: T7EI calculator

Proper assessment of gene editing with DNA mismatch detection assays

Microinjection of zebrafish embryos using Dharmacon™ Edit-R™ Cas9 Nuclease mRNA, synthetic crRNA, and tracrRNA for genome engineering

Quais as propriedades da ZsGreen1 GFP?

ZsGreen1 Fluorescent Protein

Features

References

Respondida: Quais as principais estratégias para manipulação gênica e clonagem molecular?