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. 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.