CRISPR-Cas9

This is where our journey begins. This page aims to explore the step-by-step process of manipulating and changing DNA using the revolutionary scientific technique of CRISPR-Cas9. 

We start with CRISPR, a segment of DNA containing short repeats of base sequences, associated with a protein called Cas9, which cuts the DNA (National Library of Medicine, 2022).  This mechanism uses DNA, a molecule that stores all the genetic instructions your body needs to grow, develop, and function, as well as RNA.RNA is a molecule that helps carry out the instructions in DNA. While DNA stores genetic information, RNA acts as a messenger, copying the code for a specific gene and taking it to the cell's ribosomes, where proteins are made (Wang and Farhana, 2023).

Structure :

The Cas-9 protein is a large, multi-domain DNA enzyme responsible for cutting the target DNA to form a double-stranded break and is commonly known as genetic scissors (Nishimasu et al., 2014). Cas-9 consists of two regions, called the recognition (REC) lobe and the nuclease (NUC) lobe.

The REC lobe consists of REC1 (light blue) and REC2 (green) domains responsible for binding guide RNA. Guide RNA's role is to transport the CAS9 protein to the target DNA to cut it (Bhattacharya and Satpati, 2022). 

The NUC lobe is composed: HNH (purple), PAM and PAM interacting site (Yellow), all used to ensure Cas9 binds to the correct area of DNA before it is cut. 

The process:

The mechanism of CRISPR/Cas-9 genome editing can be generally divided into three steps: recognition, cleavage, and repair. CRISPR-Cas9 targets these breaks through a distinct mechanism involving RNA guidance.

CRISPR-Cas9 gene editing follows an eight-step process (Asmamaw & Zawdie, 2021):

  • 1 Guide RNA (gRNA) Design: Identifying the specific DNA sequence to target.
  • 2 Synthesis of gRNA: Constructing the complementary RNA sequence.
  • 3 Cas9 Protein Preparation: Producing the Cas9 enzyme for DNA cleavage.
  • 4 Delivery of gRNA and Cas9 into Cells: Introducing the editing complex into target cells.
  • 5 Target Recognition: binding of gRNA to the complementary DNA sequence.
  • 6 Double-Stranded Break Formation: cleaving of the DNA at the targeted site.
  • 7 Cellular Repair: repairing the break 
  • 8 Selection and Screening: Identifying and validating successful edits.

(InVivo Biosystems, 2024)

References:

National Library of medicine (2022) What Are Genome Editing and CRISPR-Cas9?  medlineplus. Available at: https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/. (Accessed 1 April 2025)

Wang D, Farhana A. Biochemistry, RNA Structure (2023) Available from: https://www.ncbi.nlm.nih.gov/books/NBK558999/ (Accessed 7 May 2025)

Nishimasu, H., Ran, F.  Ann, Hsu, Patrick D., Konermann, S., Shehata, Soraya I., Dohmae, N., Ishitani, R., Zhang, F. and Nureki, O (2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5), pp.935–949.

Bhattacharya, S. and Satpati, P (2022) Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations. ACS Omega, 8(2), pp.1817–1837.

InVivo Biosystems (2024)HDR Vs NHEJ: DNA Repair Pathway Comparison - InVivo Biosystems. InVivo Biosystems. Available at: https://invivobiosystems.com/crispr/hdr-vs-nhej/.(Accessed 1 April 2025)

Gleditzsch, D. et al (2018) PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA biology, 16(4), pp. 504-517.

Asmamaw, M. & Zawdie, B (2021) Mechanism and Applications of CRISPR/Cas9 Mediated Genome Editing. Biologics, 15(1), pp. 353-361.

 

Images:

Chauhan, D.T (2022) Cas9 Protein: Structure, Function, Types and Importance. Genetic Education. Available at: https://geneticeducation.co.in/cas9-protein-structure-function-types-and-importance/. (Accessed 7 May 2025 )

Addgene (2015) Addgene: CRISPR Guide. Addgene.org. Available at: https://www.addgene.org/guides/crispr/.(Accessed 7 May 2025)

Uddin, F., Rudin, C.M. and Sen, T  (2020)  CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. Frontiers in Oncology, 10(1387).