Gene Editing (CRISPR/Cas9)

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.

1. Stable 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

   Documentation of monoclonality (a regulatory metric for therapeutic cell lines) is typically image-based, whereby an image of a single cell is recorded and included in regulatory filings. 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.