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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. 안정적인 Transfection

    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

    Monoclonality를 문서화(치료 세포주의 규제 지표)하는 것은 일반적으로 단일 세포의 이미지를 기록하고 이를 규제 문서 파일에 포함하는 이미지 기반 작업입니다. 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.

    응용 분야 노트 읽기  

    Clone의 생산성 Screening과 Titer

    Clone의 생산성 Screening과 Titer

    High Value Clone 식별에서 중요한 요소는 Single Cell-Derived Colony의 생산성을 결정하는 것입니다. 일반적으로 사용되는 Screening 방법은 한계희석법으로, 단일 세포를 분리하고, ELISA를 이용하여 Titer를 평가하는 여러 단계를 거치게 되어 번거롭고 시간이 오래 걸렸습니다. ClonePix 2 시스템은 표현형 선택, 단일 세포 분리 및 생산성 스크리닝을 하나의 단계로 결합하여, 스크리닝 시간을 크게 단축하고 더 많은 수의 후보물질을 다룰 수 있도록 합니다.

  • Calcein AM을 사용한 신뢰할 수 있는 클론형성능 보증

    Calcein AM을 사용한 신뢰할 수 있는 클론형성능 보증

    Viability에 대한 최소 효과를 가진 Calcein AM을 이용한 클론형성능의 확실한 보증

    여기에서는 형광 가능 CloneSelect™ Imager와 결합된 형광 시약인 Calcein AM을 사용하여 무표지 조건에 대한 유사한 Viability를 보여주면서도 동시에 높은 클론형성능을 보증해 주는 최적화된 실험과정을 살펴봅니다.

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    CRISPR/Cas9 genomic editing experiments

    CRISPR/Cas9 genomic editing experiments

    CRISPR/Cas9 유전자 편집 시스템은 비교적 사용하기 쉬우며 정확하므로 유전자 기능 연구에 많이 사용되는 도구입니다. 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.

    View poster  

  • Monoclonality

    Monoclonality assurance

    Monoclonal Antibody와 같은 생체의약품 분자를 생성하는 과정에서 Cell Line Development(세포주 개발)와 Monoclonality Assurance는 매우 중요합니다. 대상 단백질을 로버스트하게 발현하는 생존 가능한 단일 세포를 분리한 뒤, 세포주를 확립할 수 있습니다. 이 과정의 주요 이정표는 클론형성능의 근거를 문서화하는 것입니다. 클론형성능의 문서화는 일반적으로 이미지 기반으로, 단일 세포의 이미지를 생성하고 이를 규제 문서 파일에 포함합니다.

    자세히 알아보기 

    Single cell sorting

    Single cell sorting

    Cell line development 과정에는 High Value의 표적 치료 단백질을 지속적으로 생산하는 Single Cell 유래의 Clone을 찾아내는 과정이 필요합니다. 이 과정의 첫 번째 단계는 생존 가능한 단일 세포의 분리입니다. 한계희석법은 통계적 확률에 의존적인 기법이지만, 시간이 많이 소요됩니다. CloneSelect 단일 세포 프린터는 cell viability를 최대화하는 방식으로 세포를 부드럽게 분리할 뿐만 아니라 세포 분사를 통해 캡처한 5개의 이미지 시리즈로 클론형성능의 직접적인 근거를 제시할 수 있습니다.

  • Transfection 효율성

    Transfection 효율성

    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.

    응용 분야 노트 읽기  

    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.

    응용 분야 노트 읽기  

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