CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a gene editing technology that allows scientists to precisely modify DNA sequences. This innovation offers new avenues for research and potential therapeutic applications by enabling accurate targeting and alteration of genetic material.
The Essential Tools
A basic CRISPR-Cas9 protocol relies on two main components: the Cas9 enzyme and a guide RNA (gRNA). Cas9 acts as molecular scissors, cutting DNA at a precise location. The gRNA is a synthetic RNA molecule, typically around 20 nucleotides, designed to direct Cas9 to a specific target sequence within the DNA. The gRNA’s sequence is complementary to the target DNA, enabling it to bind with Cas9. For Cas9 to cut, a Protospacer Adjacent Motif (PAM) sequence must be present downstream of the target on the non-target DNA strand. The most commonly used Cas9, from Streptococcus pyogenes, recognizes an NGG PAM sequence.
The Gene Editing Journey
The gene editing process with CRISPR-Cas9 begins with target selection and guide RNA design. Researchers identify a specific DNA sequence to modify, then design a gRNA that precisely matches it, ensuring the Cas9-gRNA complex binds only to the intended genomic location. The Cas9-gRNA complex then binds to the target DNA. The Cas9 enzyme makes a double-strand break (DSB) in the DNA, typically three base pairs upstream of the PAM. This initiates the cell’s natural DNA repair mechanisms.
Cells employ two pathways to repair these double-strand breaks: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). NHEJ is a rapid, efficient, but error-prone repair pathway that ligates broken DNA ends without a homologous template, frequently resulting in small insertions or deletions (indels) that can disrupt gene function. In contrast, HDR is a more precise but less efficient repair mechanism that uses a homologous DNA template. By providing an exogenous DNA template, researchers can leverage HDR to precisely insert, correct, or replace sequences.
Getting CRISPR Inside Cells
Delivering the CRISPR components, Cas9 and gRNA, into target cells or organisms is a practical challenge.
Viral Delivery
Viral delivery methods are employed due to their high efficiency. Adeno-associated viruses (AAV) and lentiviruses are common vectors, delivering genetic instructions for Cas9 and gRNA expression into various cell types. While effective, viral vectors have limitations such as potential immunogenicity, size constraints, and difficulties in large-scale production.
Non-Viral Delivery
Non-viral approaches offer alternatives, often preferred for lower immunogenicity and capacity for larger genetic material. Electroporation uses electrical pulses to create temporary pores in cell membranes. Lipofection utilizes lipid-based reagents to encapsulate and deliver components. Microinjection, involving direct injection into individual cells, is useful for certain cell types or ex vivo applications.
Ribonucleoprotein (RNP) Delivery
RNP delivery introduces pre-assembled Cas9 protein and gRNA directly into cells. This method offers benefits such as faster action and reduced off-target effects, as components are active immediately and degrade more quickly. While promising for ex vivo applications, systemic in vivo RNP delivery is an area of active development.
Confirming the Changes
After gene editing, confirming desired changes and characterizing unintended modifications is important. Genomic DNA is extracted from edited cells and analyzed using various techniques to assess editing success.
Sanger sequencing reads the DNA sequence at the target site, confirming desired edits and identifying small insertions or deletions (indels). Next-Generation Sequencing (NGS) offers high-resolution analysis, providing precise information about nucleotide changes and detecting off-target edits across the genome. PCR-based assays, such as the T7 Endonuclease I (T7EI) assay, are used for rapid screening to detect edits by identifying mismatches in DNA strands.
Beyond DNA sequencing, functional assays confirm the biological impact of the gene edit. Observing changes in gene expression (e.g., Western blot for protein levels, qPCR for RNA levels) provides evidence of successful gene disruption or modification. Phenotypic changes in cells or organisms can also indicate successful genome editing.
Evolving CRISPR Protocols
The basic CRISPR-Cas9 protocol has undergone continuous refinement, leading to advanced gene editing techniques.
Base Editing
Base editing enables direct conversion of one DNA base to another without creating a double-strand break. For instance, cytosine base editors (CBEs) convert cytosine (C) to thymine (T), while adenine base editors (ABEs) change adenine (A) to guanine (G). This reduces the risk of unintended insertions or deletions.
Prime Editing
Prime editing allows precise insertions, deletions, and all 12 possible base conversions. This method uses a modified Cas9 enzyme fused to a reverse transcriptase, with a specialized prime editing guide RNA (pegRNA) carrying the desired edit. Prime editing offers enhanced accuracy and versatility, often without requiring a separate DNA donor template.
Deactivated Cas9 (dCas9)
Deactivated Cas9 (dCas9) expands CRISPR’s utility for gene regulation without cutting DNA. By fusing dCas9 to activator or repressor proteins, researchers create CRISPRa (CRISPR activation) or CRISPRi (CRISPR interference) systems. These systems activate or repress gene expression, providing tools for studying gene function and therapeutic applications without permanent genomic alterations.