Essential Knowledge Briefing: Genome Editing Applications for Disease Modeling and Cell Therapy
Get Started with CRISPR: From System Selection to Experimental Success
CRISPR-Cas systems have become essential tools in cell biology, enabling the precise and efficient genetic manipulation of mammalian cells. Through targeted modification of specific genes or regulatory regions, researchers can rapidly generate accurate genetic models to investigate both normal and disease-related cellular processes. In addition to their widespread use in basic research, CRISPR-based genome editing continues to show promise in therapeutic development.
This e-book offers an overview for researchers new to CRISPR or exploring its use in future experiments.
In this e-book you will find:
- A brief history of precise genome editing techniques, including CRISPR
- Guidance for choosing the right CRISPR system for your experiment
- Applications for CRISPR-Cas technology in culture systems
- Current challenges and troubleshooting
- Future directions of the field
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Page 3, Introduction
Introduction
DNA is the blueprint of life that encodes RNA and protein, as well as instructions for generation of the many different cell types that assemble into an organism. Genome editing, or the purposeful alteration of an organism’s DNA sequence, has been a long-standing goal for scientists. Efforts to carry out genome editing can be categorized into three main subtypes:
- Generation of non-specific mutations at non-specific loci. This is typically achieved by phenotype-based selection of naturally occurring genetic variants or radiation- or chemical-induced random mutagenesis.
- Insertion of specific genetic sequences into non-specific loci. The first genetically modified organism was generated through injection of preimplantation mouse blastocysts with Simian virus 40 (SV40) (1). This method allows for transgene expression from random sites in the genome where the virus DNA integrates but is incapable of precisely ‘editing’ a specific gene sequence.
- Precise genome editing at specific loci. Precise genome editing has become possible due to the discovery of naturally evolved nuclease proteins, and subsequent protein engineering efforts to harness and transform their power for a variety of applications. Precise genome editing primarily relies on the controlled induction of DNA double-strand breaks (DSBs), and their subsequent repair by endogenous DNA repair mechanisms.
This book introduces the reader to genome editing technologies, with a specific focus on the CRISPR-Cas system and how it is being successfully applied in human cells for basic and translational research.
Page 6, A Brief History Of Precise Genome Editing Technologies
Figure 1. Targeted DNA editing by CRISPR/Cas9 system.
Target DNA sequence (A) and Cas9 with corresponding guide RNA (gRNA) (B). The CRISPR-Cas9 genome editing system consists of ~100 nucleotide guide RNA (gRNA) in complex with Cas9 protein. Cas9 searches the genome for protospacer adjacent motif (PAM) sites immediately downstream of a sequence complementary to the ~20 nt protospacer/crRNA sequence within the gRNA. Cas9 will then introduce a double-strand breaks (DSB) (C and D). Cas9-induced DSBs are then repaired by either the non-homologous end joining (NHEJ) (E) or homologydirected repair (HDR) pathway, which defines editing outcome (F). Errors introduced by NHEJ can generate a variety of insertions and deletions (INDELs) that can lead to functional gene knockout. If a DNA donor template is included, the cell can use those instructions to repair the Cas9-mediated break in a precise manner via the HDR pathway (adapted from 116).
Page 8, The CRISPR Advantage
The CRISPR Advantage
Compared to previous generations of genome editing tools such as ZFNs and TALENs, several outstanding features make the CRISPR-Cas system a robust workhorse in the modern molecular biology lab:
Flexibility
The ever-expanding CRISPR toolbox includes naturally occurring and engineered Cas variants that support a variety of genetic modifications, including precise genome editing, single base editing, transcriptional activation/repression, and epigenome editing. The three main types of CRISPR systems that have been widely repurposed for genome editing (Type II, V, and VI systems) have different PAM sequence requirements; engineered Cas variants with broadened PAM specificity enable editing of a wide range of genetic loci possible.
Accessibility
CRISPR allows researchers to perform precise genome editing due to the relative ease of design and production of the requisite guide RNA sequences to target genomic sequences of interest. The expansive variety of available CRISPR tools enable cost-effective implementation and straightforward design to precisely modify the genetic or epigenetic features of target cells.
Efficiency
CRISPR outperforms traditional genome editing technologies in speed and efficacy, enabling researchers to rapidly generate genetically modified cells or organisms. Examples include ‘isogenic’ cell lines that have identical genetic information other than the disease-relevant target gene(s), and ex vivo editing of human somatic cells for therapeutic applications. CRISPR can also be used to conduct multiplex high-throughput functional screening assays by synthesizing and assembling large collections of guide RNA ‘libraries’ that can be used to systematically knock out or modulate gene expression.
Page 18, Applications Of CRISPR-Cas In Culture Systems
Applications of CRISPR-Cas Technology in Culture Systems
Genome editing has been successfully applied in numerous cell lines. While many early eukaryotic genome editing studies used immortalized cells, which are easy to culture, manipulate, and clone, more complex cells, such as stem and primary cell types, represent the most scientifically and clinically promising cell types to edit. However, genome editing of stem and primary cells has been hampered by challenges in efficient delivery and expression of the CRISPR machinery, clonogenicity, and cytotoxicity. In the coming sections, we will highlight key milestones that have enabled high efficiency editing in the most difficult-to-manipulate cells and provide example case studies that underscore the versatility and power of CRISPR-Cas genome editing when applied to cell culture systems, particularly for disease modeling and development of cellular therapies.
Next Generation Disease Modeling with CRISPR-Cas
One of the most exciting applications of CRISPR-Cas technology is its use in pluripotent stem cells (PSCs), including embryonic stem (ES) and induced pluripotent stem cells (iPSCs). PSCs have the unique capacity to expand clonally from a single cell, enabling researchers to capture relatively rare genome editing events through single-cell clonal expansion. Induced pluripotent stem cells (iPSCs) are particularly useful as they can be derived from the somatic cells of patients or healthy individuals. However, the high degree of variability between iPSC lines has presented a major challenge in using these cells to study gene function and/or disease-related phenotypes, which is at least in part due to individual genetic variation. Genome editing can circumvent this issue through the generation of isogenic clones that differ only at the genomic site-of-interest but otherwise contain an identical genetic background.
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