Was sind die effektivsten Methoden zur Quantifizierung der Effizienz der Genom-Editierung?

Polymerase Chain Reaction (PCR) involves 3 steps. They are: Denaturation: In this step, double stranded DNA gets converted to Single stranded DNA. This step takes place at 94-95 degree celcius. Annealing: Primers get attached at the end of the strands. This step takes place at 50-56 degree celcius. Extension: This step takes place at 72 degree celcius where taq polymerase adds nucleotides to the annealed primers.

To be straightforward, the absence of a universally recognized and readily available "gold standard" method further complicates the challenges encountered by the genomics scientific community. In recent attempts at CRISPR-Cas9 genome editing, we've made notable advancements by enhancing precision and minimizing off-target effects. However, most genome editing studies mainly look at tiny changes called INDELs, which result from fixing double-strand breaks. They indeed typically use simple PCR-based methods (and/or digital PCR) for this analysis.

PCR-Based Methods: PCR assays, such as T7 endonuclease I (T7EI) or high-resolution melting (HRM) analysis, detect mismatches created by genome editing tools like CRISPR-Cas9. These methods assess indel formation by measuring cleavage efficiency. Sequencing-Based Methods: Amplicon or target region sequencing allows direct identification and quantification of edited sequences, providing precise measurement of editing efficiency and types of edits generated. Fluorescence-Based Assays: Fluorescent reporters or assays like TIDE (Tracking of Indels by Decomposition) use fluorescent tags to measure editing outcomes, enabling rapid evaluation of editing efficiency.

1. Secuenciación de nueva generación (NGS): Permite detectar las modificaciones genéticas introducidas y evaluar la eficacia de la edición del genoma. 2. Análisis de PCR cuantitativo (qPCR): Se puede utilizar para medir la frecuencia de edición génica y determinar la eficacia de la edición en células o tejidos. 3. Secuenciación de un solo genoma (SGS): Permite analizar genomas individuales para evaluar la eficacia y precisión de las ediciones introducidas. 4. Ensayos de clonaje y secuenciación: Se emplean para identificar y cuantificar las ediciones específicas introducidas.

I recommend leveraging Long-read sequencing technology for its capacity to generate reads spanning a broad range, from short to several thousand kilobases. This capability proves critical in detecting significant structural variations within the genome, particularly those that elude detection through PCR methods. Noteworthy applications include the use of Pacific Biosciences' single-molecule real-time sequencing (SMRT-seq) and Oxford Nanopore Technologies' Nanopore sequencing for the precise quantification of gene modifications (induced by CRISPR-Cas9).

Sequencing based method or commonly known as Next Generation Sequencing involves four steps. They are: Extraction and isolation of DNA: The DNA is extracted from a relevant source. Library preparation: The sample is fragmented into equal sized targets. Then adapters are added at both ends and are allowed to interact at NGS platform. A library involves a collection of molecules that can be sequenced. Clonal Amplification and Sequencing: In clonal amplification, DNA fragments are amplified for sequencing. This is done by adding beads. Sequencing by synthesis (SBS) involves loading of library onto the sequencer which then reads the nucleotides one by one. Data Analysis: This step involves processing, analysis and interpretation.

1. Secuenciación de nueva generación (NGS): Permite detectar las modificaciones genéticas introducidas y evaluar la eficacia de la edición del genoma. 2. Análisis de PCR cuantitativo (qPCR): Se puede utilizar para medir la frecuencia de edición génica y determinar la eficacia de la edición en células o tejidos. 3. Secuenciación de un solo genoma (SGS): Permite analizar genomas individuales para evaluar la eficacia y precisión de las ediciones introducidas. 4. Ensayos de clonaje y secuenciación: Se emplean para identificar y cuantificar las ediciones específicas introducidas en clones celulares.

Computational method is yet another technique to quantify genome editing efficiency. This method involves use of computational tools like CRISPR.

A thorough comprehension and accurate measurement of various gene editing results, encompassing deletions (LDs) and substantial insertions, are crucial for strategically designing, functionally analyzing, and effectively applying CRISPR-Cas9–based genome editing. This quite detailed characterization is instrumental, not only in enhancing the overall effectiveness but specifically in ensuring the precision of CRISPR-Cas9 technology in genome editing procedures.