A Technical Primer on CRISPR-Cas9 Gene Editing

CRISPR-Cas9 technology has revolutionized the field of molecular biology by providing a simple yet powerful tool for precise genome editing. In this blog post, we'll delve into the technical details of CRISPR-Cas9 and its applications in genetic engineering.

Understanding CRISPR-Cas9

CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9. It is a genome editing system derived from the bacterial immune system, specifically the adaptive immune response of certain bacteria to viral infections.

Components of CRISPR-Cas9

  1. CRISPR Array: The CRISPR array consists of short, repetitive DNA sequences interspersed with unique spacer sequences derived from previous encounters with foreign genetic elements, such as viruses or plasmids.
  2. Cas9 Protein: Cas9 is an RNA-guided endonuclease enzyme that is responsible for cleaving DNA at specific target sequences. It forms a complex with a single guide RNA (sgRNA) molecule, which directs it to the complementary target sequence in the genome.
  3. Single Guide RNA (sgRNA): The sgRNA is a synthetic RNA molecule that consists of a CRISPR RNA (crRNA) sequence, which recognizes the target DNA sequence, and a trans-activating crRNA (tracrRNA) sequence, which binds to the Cas9 protein and facilitates its function.

abm | saCas9 Null Mutant Protein | K146

Mechanism of Action

  1. Recognition: The sgRNA binds to the target DNA sequence through complementary base pairing, guiding the Cas9 protein to the specific genomic locus.
  2. DNA Cleavage: Once bound to the target sequence, Cas9 induces a double-stranded break (DSB) in the DNA by cleaving both strands of the double helix.
  3. Repair: The cell's DNA repair machinery then repairs the DSB through one of two main pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in small insertions or deletions (indels) at the site of the break, while HDR can be used to introduce specific genetic changes by providing a donor DNA template.

Applications of CRISPR-Cas9

  1. Gene Knockout: CRISPR-Cas9 can be used to disrupt or "knock out" specific genes by introducing indels at target loci, thereby disrupting gene function.
  2. Gene Editing: It enables precise editing of DNA sequences by providing a template for HDR-mediated repair, allowing for the introduction of specific genetic changes, such as point mutations or insertions.
  3. Gene Regulation: CRISPR-Cas9 systems have been adapted for transcriptional regulation by fusing the catalytically inactive form of Cas9 (dCas9) with transcriptional activators or repressors, allowing for precise control of gene expression.

Plasmid-encoded CRISPR-Cas systems mediate complex plasmid-MGE interactions


CRISPR-Cas9 technology has opened up new possibilities for manipulating the genome with unprecedented precision and efficiency. Its simplicity, versatility, and affordability have made it a widely adopted tool in basic research, biotechnology, and potential therapeutic applications. Understanding the technical aspects of CRISPR-Cas9 is essential for harnessing its full potential and advancing scientific knowledge and innovation.

5th Apr 2024 Wiem Gasri

Recent Posts