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. 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.
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.
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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.
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.
Clone productivity screening and titer
An important component in identifying high-value clones is determining productivity of single cell-derived colonies. Screening for productivity using traditional approaches is laborious and time consuming, generally consisting of a multistep process that involves isolating single cells from limiting dilution followed by assessment of titer using ELISA. The ClonePix 2 system combines phenotype selection, single-cell isolation and productivity screening into a single step, resulting in dramatically shorter screening times and increased number of candidates.
Clone productivity screening and titer
An important component in identifying high-value clones is determining productivity of single cell-derived colonies. Screening for productivity using traditional approaches is laborious and time consuming, generally consisting of a multistep process that involves isolating single cells from limiting dilution followed by assessment of titer using ELISA. The ClonePix 2 system combines phenotype selection, single-cell isolation and productivity screening into a single step, resulting in dramatically shorter screening times and increased number of candidates.
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Confident assurance of clonality using calcein AM
Confident assurance of clonality using calcein AM with minimal effect on viability
Here, we demonstrate an optimized workflow using the fluorescence reagent, calcein AM, in conjunction with a fluorescence-capable CloneSelect™ Imager that shows similar viability to label-free conditions while simultaneously providing high assurance of clonality.
Confident assurance of clonality using calcein AM
Confident assurance of clonality using calcein AM with minimal effect on viability
Here, we demonstrate an optimized workflow using the fluorescence reagent, calcein AM, in conjunction with a fluorescence-capable CloneSelect™ Imager that shows similar viability to label-free conditions while simultaneously providing high assurance of clonality.
CRISPR/Cas9 genomic editing experiments
The CRISPR/Cas9 gene editing system is a very popular tool for studying gene function due to its relative ease of use and accuracy. 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.
CRISPR/Cas9 genomic editing experiments
The CRISPR/Cas9 gene editing system is a very popular tool for studying gene function due to its relative ease of use and accuracy. 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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Monoclonality
Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies. A cell line can be established following the isolation of a single viable cell robustly expressing the protein of interest. A key milestone in this process is documenting evidence of clonality. Documentation of clonality is typically image-based, whereby an image of a single cell is produced and included in regulatory filings.
Monoclonality
Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies. A cell line can be established following the isolation of a single viable cell robustly expressing the protein of interest. A key milestone in this process is documenting evidence of clonality. Documentation of clonality is typically image-based, whereby an image of a single cell is produced and included in regulatory filings.
Single cell sorting
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. The first step in the process is the isolation of single, viable cells. Limiting dilution is a technique that relies on statistical probability but is time consuming. The CloneSelect Single-Cell Printer enables the gentle isolation of cells in a manner that maximizes cell viability, as well as providing direct evidence of clonality via a series of five images captured as cells dispense.
- A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
- A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
- Data Sheet: CloneSelect Single-Cell Printer
Single cell sorting
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. The first step in the process is the isolation of single, viable cells. Limiting dilution is a technique that relies on statistical probability but is time consuming. The CloneSelect Single-Cell Printer enables the gentle isolation of cells in a manner that maximizes cell viability, as well as providing direct evidence of clonality via a series of five images captured as cells dispense.
- A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
- A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
- Data Sheet: CloneSelect Single-Cell Printer
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Transfection efficiency
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.
Transfection efficiency
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.
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.
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.
Latest Resources
Resources of Gene Editing
eBook
How to Implement Colony Picking Workflows with Automation
How to Implement Colony Picking Workflows with Automation
Colony picking represents a powerful tool within the workflow of mammalian cell line development and the study of microbiology.
Application Note
CloneSelect Imager FL fluorescence for rapid day zero monoclonality
CloneSelect Imager FL fluorescence for rapid day zero monoclonality
Gene editing allows DNA manipulation, i.e., addition, deletion, or modification using different tools. Several nucleases for genome editing are now available: zinc finger nucleases (ZFN),…
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Application Note
A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
In this app note, we demonstrate a unified workflow that combines single cell deposition and clonality verification. We couple the use of the SCP and CSI to deposit single cells into a…
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Application Note
A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
The isolation of single cells has a broad spectrum of applications, from single-cell genomics to antibody discovery and cell line development.
Scientific Poster
Validating Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 Genomic Editing Experiments Using Molecular Devices Instruments
Validating Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 Genomic Editing Experiments Using Molecular Devices Instruments
The CRISPR/Cas9 gene editing system is a very popular tool for studying gene function due to its relative ease of use and accuracy. Additionally, the system has enormous potential for treati…
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Application Note
Validate CRISPR-Edited Cells using Imaging and Western Blot Detection on a Microplate Reader
Validate CRISPR-Edited Cells using Imaging and Western Blot Detection on a Microplate Reader
Genome editing has been widely used to study gene expression and protein function, but many of these methods are labor intensive and inaccurate. The CRISPR (Clustered Regularly Interspaced…
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Application Note
Confident assurance of clonality using calcein AM with minimal effect on viability
Confident assurance of clonality using calcein AM with minimal effect on viability
The development of cell lines that express a specified protein of interest is critical to the generation of biopharmaceutical molecules such as monoclonal antibodies. To establish a cell…
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Scientific Poster
Rapid Screening and Selection of Stable High Producing Clones
Rapid Screening and Selection of Stable High Producing Clones
Screening cell populations for stable, high producing clones is a key rate-limiting step in the development and production of new protein therapeutics.
Data Sheet
CloneSelect Single-Cell Printer Series
CloneSelect Single-Cell Printer Series
The CloneSelect™ Single-Cell Printer™ Series deposit single cells gently and with high efficiency using a patented, inkjet-like disposable, oneway dispensing cartridge.
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Application Note
Automated Optimization of IgG Production in CHO Cells
Automated Optimization of IgG Production in CHO Cells
Monoclonal antibodies have long been an essential tool for cell and molecular assays and have moved into the clinic as part of a shift from small molecules into biologic treatments for…
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Application Note
Enhanced development of virus-specific hybridomas using ClonePix and CloneSelect Imager Technologies
Enhanced development of virus-specific hybridomas using ClonePix and CloneSelect Imager Technologies
The ClonePix System was employed in the high-throughput screening of hundreds of sub-cloned colonies from parental hybridoma material (historically parental line yields were less than 1 mg/…
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Application Note
Optimizing GFP transfection with the SpectraMax i3 Multi-Mode Detection Platform
Optimizing GFP transfection with the SpectraMax i3 Multi-Mode Detection Platform
Expression of fluorescent proteins, for example, green fluorescent proteins (GFP), is often used as a read-out for reporter gene assays. In a typical reporter assay, the regulatory sequence…
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