CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, is a gene-editing technology. Originally discovered as a bacterial immune defense, this system allows scientists to precisely modify DNA sequences in virtually any organism. It operates like molecular scissors, enabling targeted cuts to the genetic code. CRISPR has made gene editing simpler, more affordable, and more accurate than previous methods, transforming biomedical research.
Selecting the Target and Designing the Guide
The initial step in a CRISPR experiment involves identifying the specific DNA sequence for modification, known as the target. Scientists then design a guide RNA (gRNA), a short RNA molecule complementary to this target DNA. This complementarity ensures the gRNA precisely binds to the desired genomic location.
The gRNA has two main parts: a 20-nucleotide “spacer” (crRNA) for sequence-specific targeting, and a “scaffold” (tracrRNA) that binds to the Cas9 protein. The target sequence must be immediately upstream of a protospacer adjacent motif (PAM) site, a short DNA sequence recognized by Cas9. Designing a gRNA with high specificity and minimal off-target binding is important for accurate gene editing.
Getting CRISPR into Cells
Once prepared, the Cas9 enzyme and guide RNA must be delivered into target cells. This delivery uses various methods, broadly categorized as viral and non-viral approaches. Viral vectors, such as adeno-associated viruses (AAVs) and integrase-defective lentiviral vectors (IDLVs), leverage viruses’ natural ability to infect cells and introduce genetic material. AAVs are often favored for in vivo applications due to their low immunogenicity and persistence in non-dividing cells, making them suitable for tissues like the liver and brain.
Non-viral methods offer alternatives with reduced immunogenicity and greater customization. Lipid nanoparticles (LNPs) are a prominent system, encapsulating and protecting CRISPR components like mRNA or ribonucleoproteins (RNPs). These nanoparticles facilitate cellular uptake and protect the cargo from degradation, proving effective for liver-targeted treatments. Other non-viral techniques include electroporation, which uses electrical pulses to create temporary pores in cell membranes, and polymeric nanoparticles, known for biocompatibility and biodegradability.
How DNA is Edited
After the CRISPR components enter the cell, the guide RNA directs the Cas9 enzyme to the precise target DNA sequence. The gRNA’s complementary “spacer” region base-pairs with the target DNA, positioning Cas9 accurately. Upon successful binding, the Cas9 enzyme then acts as a molecular scissor, creating a double-strand break (DSB) in the DNA molecule at the specified location. This break is the initial event that triggers the cell’s natural DNA repair mechanisms.
The cell primarily employs two repair pathways to mend these DSBs: Non-Homologous End Joining (NHEJ) and Homology-Directed Repair (HDR). NHEJ is an “error-prone” pathway that quickly rejoins broken DNA ends without a homologous template. This process often results in small insertions or deletions (indels) at the repair site, which can disrupt gene function and lead to gene knockout. NHEJ is active throughout the cell cycle and is the cell’s preferred repair mechanism.
In contrast, HDR is a more precise repair pathway that utilizes a homologous DNA template to accurately fix the DSB. This template can be a sister chromatid during cell division or an exogenously supplied DNA sequence provided by researchers. HDR allows for specific genetic modifications, such as introducing precise point mutations, correcting existing mutations, or inserting new DNA sequences (gene knock-ins). While HDR is highly accurate, it is less efficient than NHEJ and primarily active during the S/G2 phases of the cell cycle, when a homologous template is available. Researchers can manipulate these pathways to achieve desired editing outcomes, often by providing a donor template to encourage HDR or by inhibiting NHEJ to increase HDR efficiency.
Verifying the Edit
Following gene editing, confirming the desired genetic modification is a final step. This verification ensures the accuracy and success of the CRISPR experiment. Scientists often observe phenotypic changes in edited cells as a preliminary indicator, such as the activation or deactivation of a fluorescent protein. However, more precise molecular methods are required to confirm exact DNA sequence alterations.
Common verification techniques include PCR-based assays and DNA sequencing. Enzymatic mismatch cleavage assays, like those using T7E1 or Surveyor nucleases, can detect the presence of insertions or deletions by cleaving mismatched DNA strands that form when edited and unedited DNA are hybridized. While these methods offer quick assessments, they do not provide specific sequence information. For detailed analysis, Sanger sequencing or Next-Generation Sequencing (NGS) are employed, which directly determine the DNA sequence at the target site and can identify specific nucleotide changes, including single-nucleotide substitutions and off-target edits.