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Gene Editing (CRISPR/Cas9)

Automated solutions to scale up the promise of gene editing with CRISPR engineering

What is gene editing?

Gene editing is a genetic manipulation in which a living organism’s genomic DNA is deleted, inserted, replaced, or modified. Gene editing is a site-specific targeting to create breaks in DNA through various techniques and does not always involve repair mechanisms. It consists of two techniques – inactivation and correction.

Inactivation involves the turning of a target gene, and correction facilitates the repair of the defective gene through a break in the gene. Gene editing has vast potential in a myriad of fields, including drug development, gene surgery, animal models, disease investigation and treatment, food, biofuel, biomaterial synthesis, and others.

Though CRISPR, a major gene editing technique, has been extensively used recently, gene editing was first studied in the late 1900s. Since the onset of CRISPR, previously an ambitious application, gene therapy has become the most sought-after application of gene editing. This can be achieved through two approaches, gene addition, which adds to the existing genetic material to make up for faulty or missing genes, and gene editing, which treats diseases by directly modifying the disease-related DNA.

CRISPR/Cas9 Mechanism

CRISPR/Cas9 Mechanism. The Cas9 enzyme is activated by first binding to a guide RNA, then binding to the matching genomic sequence that immediately precedes 3-nucleotide PAM sequence. The Cas9 enzyme then creates a double-strand break, and either the NHEJ or the HDR pathway is used to repair the DNA, resulting in an edited gene sequence.

A guide RNA (gRNA) similar to a crRNA is designed to target a region in the gene, and the Cas9 enzyme can create doublestrand breaks in this specific region of the host cell’s genome (Figure 1). After a double-strand break is made, the cell will undergo one of two repair pathways: the nonhomologous end joining (NHEJ) pathway or the homology-directed recombination (HDR) pathway. The NHEJ pathway is commonly used to disrupt genes via base insertions or deletions (indels), while the HDR pathway can be used to knock in a reporter gene or an edited sequence by exchanging sequences between two similar or identical molecules of DNA.

 

Scaling up gene editing with CRISPR engineering

“CRISPR” – Clustered Regularly Interspaced Short Palindromic Repeats. These DNA sequences were first discovered as a part of immune system in prokaryotes such as bacteria and archaea, and garnered importance as a gene editing tool since 2012 (Jinek et al., 2012). It has a great promise in a myriad of applications, i.e. including, agriculture, disease modeling, gene therapy, drug discovery to name a few. The precision it has makes it a perfect tool for insertion (knock-ins), deletion (knockouts) and other modifications of DNA sequences. It has replaced existing tedious and expensive gene-editing tools like TALENS and ZFNS to a large extent.

CRISPR sequences contain DNA from previous viral invaders called spacers after each palindromic repeat, and these aid in detection and destruction of similar future viruses. Understanding this mechanism (Jinek et al., 2012) led to the first use of CRISPR in eukaryotic cells (Cong, L, et al., 2013) and later in other cell types plus organisms pertaining to different fields. The CRISPR – Cas9 systems has two major components which form a ribonucleoprotein complex. The first component or guide RNA binds to a complementary DNA sequence in genome and the second component Cas9 from Streptococcus pyogenes (SpCas9) makes a double strand break at the site of target. A protospacer adjacent motif (PAM) is where the nuclease initially binds for the upstream cut to occur. Different CRISPR nucleases have different PAM sites and once the cut is made the cells repair system is activated and edits to the genome is initiated as well.

Gene editing workflow

Gene editing workflow using CRISPR mechanisms to attain a confirmed edit cell line has various steps. Effective optimization of these steps using the right tools contributes to an efficient process to cut down the time, effort, and costs of various scientific advances. This approach helps accelerate R&D, revolutionizes drug discovery, disease cure, gene-edited crop production, etc. We discuss the steps involved and effective solutions we offer to support the scientific communities worldwide to achieve their endeavors through gene-editing.

Gene editing workflow

 

  1. Stabile Transfektion

    Identification of the best method for delivering the CRISPR-Cas9 system into the cells of interest is the first step in the gene editing workflow. When considering which transfection method to use, transfer efficiency and subsequent cell viability are important factors. Transfection efficiency optimization, construct design, delivery method assessment, host line selection are some important factors to be considered.

  2. Pool generation and expansion

    Creating a custom gene-modified cell line starts with the evaluation of the transfected cell pool to effectively screen the edited from the unedited in using different selection methods like antibiotic based, fluorescent protein reporter based, antibody tagged cell sorting, and others. The successfully transfected/screened cell pool is then expanded for further monoclonal cell line development.

  3. Enrichment & single-cell isolation

    Enrichment for cells of interest occurs after cells have been transfected. In this step, only those cells that carry the desired edits are identified and expanded. Individual cells are then isolated for confirmation of monoclonality required for regulatory approval.

  4. Monoclonality verification and growth

    Die Dokumentation der Monoklonalität (eine regulatorische Vorschrift für therapeutische Zelllinien) basiert üblicherweise auf Abbildungen, wobei die Aufnahme einer einzelnen Zelle erfasst und in die Zulassungsunterlagen mit aufgenommen wird. Many researchers now routinely use imaging systems, such as the CloneSelect Imager, to verify monoclonality at day 0, and monitor cell growth in cell culture media.

  5. Verification and functional confirmation of edits

    It is important to confirm that target cells have been successfully edited prior to moving on to downstream assays. This can be accomplished either through direct detection of edits using genomic methods or through indirect detection using cellular or proteomic methods. Picking an appropriate assay for your system is the key. Downstream assays for verification and functional confirmation could be picked from various conventional/ NGS methods. Conventional : PCR, Sanger, qPCR, western blot, cell – based assays, etc. NGS : High resolution on and off –target assessment, Single cell RNA- seq, ChIP-Seq, etc

  6. Scale up for applications - Analyze and make discoveries

    Phenotype investigation can begin once it has been confirmed that the cells are correctly edited. Further evaluation of the system with a drug as part of a cell-based assay may be desired during target or lead discovery and validation.

Research solutions for validating CRISPR/Cas9 gene edits

Molecular Devices’ family of instruments can effectively be used to perform/screen experiments ensuring the success of gene-editing endeavors. The new CloneSelect Imager Florescence (CSI-FL) provides monoclonality Day0 assurance after single-cell printing, transfection efficiency, cell confluency, and multichannel fluorescence screening data to validate gene editing efficacy through shorter tracking times, low risk of over passaging, and robotics. 

In addition, our SpectraMax i3x Multi-Mode Microplate Reader can be used to assess transfection efficiency, monitor cell growth, quantitate DNA & protein, and validate CRISPR/Cas9 edits through ScanLater Western Blot analysis. High-quality images of autophagosomes can be acquired using the ImageXpress Micro Confocal System while the MetaXpress HCI software can identify and quantitate individual autophagosomes from every cell allowing us to analyze phenotypic changes occurring from the CRISPR/Cas9 gene edits.

  • Accelerating gene edited cell lines

    Accelerating gene edited cell lines

    Learn how the all new CloneSelect® Imager FL can aid in easy detection of successfully transfected cells, cutting cell line development timelines and scaling up your research faster. Reject low transfection efficiency pools at an early stage, confirm and track various CRISPR edits with multi-channel fluorescence detection, and screen cells with accuracy and confidence while reducing the risk of over-passing disturbances with robotics redesign.

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    Screening auf Produktivität von Klonen und Titer

    Screening auf Produktivität von Klonen und Titer

    Eine wichtige Komponente in der Identifizierung hochwertiger Klone ist die Bestimmung der Produktivität der aus einer einzigen Zelle gewonnenen Kolonie. Das Screening auf Produktivität mit traditionellen Ansätzen ist arbeits- und zeitintensiv. Allgemein besteht der Prozess aus mehreren Schritten, darunter die Isolation einzelner Zellen aus limitierenden Verdünnungsreihen, gefolgt von der Bestimmung des Titers mittels ELISA. Das ClonePix 2 System vereinigt die Selektion von Phänotypen, die Isolation von Einzelzellen und das Produktivität-Screening in einem einzigen Schritt, wodurch erheblich kürzere Screening-Zeiten und eine höhere Anzahl an Kandidaten erzielt werden.

  • Zuverlässige Sicherstellung der Klonalität mit Calcein-AM

    Zuverlässige Sicherstellung der Klonalität mit Calcein-AM

    Zuverlässige Sicherstellung der Klonalität durch Verwendung von Calcein-AM mit minimalen Auswirkungen auf die Überlebensfähigkeit

    Hier präsentieren wir einen optimierten Arbeitsablauf zur Verwendung des fluoreszenten Reagenz Calcein-AM zusammen mit einem Fluoreszenz-fähigen CloneSelect™ Imager. Er weist eine ähnliche Überlebensfähigkeit der Zellen auf wie unter labelfreien Bedingungen, während er gleichzeitig mit hoher Sicherheit einen Klonitätsnachweis liefert.

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    CRISPR/Cas9 genomic editing experiments

    CRISPR/Cas9 genomic editing experiments

    Das CRISPR/Cas9-Genediting-System ist ein sehr populäres Werkzeug zur Untersuchung der Genfunktion, da es relativ leicht anzuwenden und sehr genau ist. Additionally, the system has enormous potential for treating hereditary diseases. Validation of CRISPR/Cas9 gene editing is necessary to ensure that genes of interest are successfully knocked down or knocked out. Here, we demonstrate how Molecular Devices' family of instruments can be utilized in gene editing experiments by using CRISPR/Cas9 to knockdown autophagy-related protein 5 (ATG5) in HEK293 cells.

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  • Monoklonalität

    Sicherstellung der Monoklonalität

    Die Entwicklung von Zelllinien und die Sicherstellung der Monoklonalität sind entscheidende Schritte im Herstellungsprozess biopharmazeutischer Moleküle, wie z. B. monoklonaler Antikörper. Eine Zelllinie kann nach der Isolierung einer einzigen überlebensfähigen Zelle, die das Protein von Interesse robust exprimiert, etabliert werden. Ein entscheidender Meilenstein in diesem Prozess ist die Dokumentation des Nachweises der Klonalität. Die Dokumentation der Klonalität basiert üblicherweise auf Abbildungen, wobei eine Aufnahme einer einzelnen Zelle erstellt und in die Zulassungsunterlagen mit aufgenommen wird.

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    Einzelzellsortierung

    Einzelzellsortierung

    Die Entwicklung von Zelllinien erfordert das Auffinden einzelner aus Zellen hervorgegangener Klone, die hohe und gleichbleibende Mengen des therapeutischen Zielproteins herstellen. Der erste Schritt in diesem Prozess ist die Isolierung einzelner, überlebensfähiger Zellen. Die limitierende Verdünnungsreihe ist eine Technik, die sich auf statistische Wahrscheinlichkeit stützt, jedoch zeitraubend ist. Der CloneSelect Single-Cell Printer ermöglicht eine schonende Isolierung von Zellen auf eine Art und Weise, die die Überlebensfähigkeit von Zellen maximiert, und dabei auch einen direkten Nachweis der Klonalität durch eine Serie von fünf Bildern, die während des Absetzens der Zellen aufgenommen werden, bietet.

  • Transfektionseffizienz

    Transfektionseffizienz

    Transfection efficiency for a fluorescent reporter gene can be monitored in different ways. One way is to measure fluorescence with a microplate reader. This allows one to assess the overall fluorescence level in each test well, but it does not give the percent of cells transfected. A more informative way to assess transfection efficiency is to analyze the cells using an imaging cytometer, where the number of cells expressing detectable fluorescence can be compared to the total cell number. The imaging cytometer has the added benefit of enabling calculation of cell confluence prior to transfection, so that this information can be used as part of assay development.

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    Validate CRISPR-Edited Cells using Western Blot

    Validate CRISPR-Edited Cells using Western Blot

    CRISPR gene-editing technology requires careful monitoring of the entire process to ensure accurate results. The SpectraMax i3x Multi-Mode Microplate Reader provides a complete solution for analyzing the results of a CRISPR-editing experiment from initial transfection to confirmation of protein knockdown. With the MiniMax cytometer, researchers can assess transfection efficiency by comparing total unlabeled cell counts to counts of fluorescence expressing transfected cells. The ScanLater Western Blot Detection System enables sensitive detection and quantitative analysis of proteins of interest in control and CRISPR-edited cells.

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