CRISPR-Cas9 Genome Editing

The CRISPR–Cas9 Genome Editing System

Genome editing is a gene therapy strategy that utilizes engineered or bacterial nucleases to deliver targeted changes in the genome to prevent or treat a disease.1,2 In this video, we will show you how genome editing can be initiated by the CRISPR–Cas9 system. CRISPR stands for clustered regularly interspaced short palindromic repeats, and Cas9 stands for CRISPR-associated protein 9.

CRISPR–Cas9 is an RNA–protein-based system that facilitates RNA-guided site-specific DNA cleavage.3,4 CRISPR–Cas9 is one of the more widely investigated programmable endonucleases used for genome editing.5,6

In October 2020, Dr. Charpentier and Dr. Doudna were awarded The Nobel Prize in Chemistry for their discovery of the CRISPR–Cas9 system as a tool for genome editing.7

The CRISPR–Cas9 system is derived from the CRISPR–Cas adaptive immune system in bacteria. Bacteria use this system to degrade foreign genetic materials from invading viruses and plasmids via three steps.8,9

In the first step, called adaptation, a segment of the invading viral DNA is integrated into a part of the bacterial chromosome called the CRISPR locus.9,10 The CRISPR locus is an array of short repeated sequences alternated with spacers that are often derived from genetic material of invading viruses and plasmids.11 In the second step, called expression, the CRISPR locus containing the foreign DNA gets transcribed and then processed to a short RNA called the RNA guide. This RNA guide contains a sequence of approximately 20 nucleotides that is complementary to the invading foreign DNA.9,10,12 In the last step, interference, the newly transcribed RNA guide interacts with a type of enzyme called Cas that cleaves genetic material. The RNA guide then directs Cas to the complementary foreign DNA for cleavage.9,10,12 The RNA guide binds to the complementary site in the invading foreign DNA, and the Cas protein cleaves the foreign genetic material, degrading it.9,10

The CRISPR–Cas9 system used for genome editing consists of two components — an RNA guide strand and the Cas9 endonuclease.5 The RNA guide and Cas9 interact to form an active DNA surveillance complex.12 The RNA guide provides target site recognition and can be designed to target almost any DNA sequence in the genome to introduce site-specific changes in the DNA.5,12 The Cas9 endonuclease is a large multidomain protein that cleaves DNA.12

The RNA guide is a single chimeric RNA consisting of two RNA pieces that have distinct functions.8,13 One piece of the RNA guide contains the targeting guide sequence, an approximately 20-nucleotide-long sequence that is complementary to a part of the target DNA called the protospacer sequence.8,13 Within this 20-nucleotide sequence is a shorter seed region that is important for Cas9 specificity for the target sequence and efficient cleavage.8,13 The other piece in the RNA guide, shown in dark green, mediates recognition of the target DNA sequence and is required for Cas9 recruitment and Cas9-mediated cleavage of the target.8,12

Cas9 interacts with the target DNA via a sequence in the target DNA called the protospacer adjacent motif or PAM sequence.5,8,13 The PAM sequence is adjacent to the protospacer sequence and is critical for Cas9-specific recognition of the target DNA sequence.5,8,14 The RNA guide–Cas 9 complex binds to and unwinds the DNA, searching for the target sequence to which the RNA guide has complementarity.15

Once the Cas9 endonuclease interacts with the PAM sequence, the DNA strands separate out, further allowing the RNA guide to bind to the protospacer sequence that is immediately upstream to PAM.14 Following this, Cas9 induces a break in the double-stranded DNA at about 3 base pairs upstream to the PAM sequence.8,12 This break in DNA activates the endogenous DNA repair pathways that can be used to induce targeted modifications in the host genome by either nonhomologous end joining or homology-directed repair.5

This CRISPR–Cas9 system is used for introducing changes in the genome in a spectrum of organisms due to its ease of design, simplicity in use and high efficiency.12 It can be delivered in a system such as a plasmid or viral vector.16,17

Although still in development, CRISPR–Cas9 is being investigated to correct or eliminate mutations associated with cancer, sickle cell anemia, beta-thalassemia, retinitis pigmentosa, Leber congenital amaurosis and cardiovascular disease.1,18

The CRISPR–Cas9 system is also being used in cancer immunotherapy.1,18 Using CRISPR–Cas9, isolated T cells are engineered to express chimeric antigen receptors or CARs.1,18 These genetically engineered CAR T cells can recognize and kill tumor cells specified by the CAR.1,18

In summary, CRISPR–Cas9 is a powerful genome editing tool that can be used to induce sequence-specific changes in the DNA and is actively being investigated as a therapeutic strategy in a variety of diseases.1,12


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