Some bacteria have a system for recognizing an invading virus and destroying it. The system is called CRISPR (for clustered regularly interspaced short palindromic repeats) [Link to a discussion of palindromes.] It is described on a separate page — Link to it.
Like restriction enzymes (also discovered in bacteria), CRISPR provides a way of targeting a particular DNA sequence. The CRISPR gene encodes an RNA (crRNA) that contains a length of 23 nucleotides that will bind by Watson-Crick base-pairing to the complementary sequence on a strand of DNA.
The CRISPR RNA molecule also binds to an endonuclease called Cas9 that cuts both strands of the DNA within the target sequence.
Although found in bacteria, the CRISPR-Cas9 system can be introduced into eukaryotic cells where it enables specific genes to be altered.
Example:
The CRISPR-Cas9 genes can be introduced into a eukaryotic cell on a plasmid. Expression of the CRISPR RNA (crRNA) and the Cas9 protein cuts both strands of the target DNA sequence. When the host DNA repair enzymes repair the double-stranded break (DSB) by NonHomologous End-Joining (NHEJ), this error-prone process is apt to introduce insertions or deletions (indels) into the DNA sequence. Often such indels will create a frameshift thus inactivating the gene.
Another Example:
One can introduce a plasmid containing the CRISPR-Cas9 genes AND a gene that one hopes to incorporate into the host cell's genome. If the double-stranded break is repaired by homologous recombination (homology-directed repair), the introduced gene can be inserted into the site.
Genome editing with the CRISPR-Cas9 system has worked successfully in a variety of microbes, plants, nonhuman animals, and now (2023) humans.
The first genome editing procedure to be used therapeutically in humans is expected to be approved in the U.S. in late 2023 or early 2024.
It is a treatment for sickle cell disease (but can serve as well for beta-thalassemia). Sickle cell disease is caused by a mutation in the genes encoding the beta chain of hemoglobin. [LINK to a discussion].
The treatment involves infusing the patient with their own blood-forming cells that, using CRISPR-Cas9 technology, have been treated to enhance the synthesis of fetal hemoglobin. Fetal hemoglobin uses two gamma chains instead of the beta chains of adult hemoglobin (the ones defective in sickle cell disease). Red cells containing sufficient fetal hemoglobin do not sickle. [Link for more details]
Gene targeting with CRISPR-Cas9 cuts both strands of the DNA. Repair of the break can introduce harmful changes in the sequence.
There is a modified version of CRISPR that cuts only one strand of the DNA. In the process, a single base can be altered. For example, swapping the guanine base (G) for adenine (A) in the PCSK9 gene disables it. This is valuable because the PCSK9 protein increases the concentration of low-density lipoproteins (LDLs, "bad" cholesterol) in the circulation. Disabling the gene prevents this.
PCSK9 is a serine protease which binds to the complex of LDL and its receptor (LDLR) promoting the degradation of the receptor within the cell. With fewer LDL receptors returning to the cell surface [View] to remove more LDLs, the level of LDLs in the blood rises. By blocking the synthesis of PCSK9 within the cell (chiefly liver cells), the concentration of "bad" cholesterol in the blood is lowered potentially reducing the risk of heart attacks and other cardiovascular problems.
Clinical trials of a single injection of this base editing construct show promising results.
On March 10, 2025, Beam Therapeutics announced the results of treating 9 patients with alpha-1-antitrypsin. LINK to a description of the disease.
The treatment consisted of a single dose of a modified CRISPR that entered the liver cells of the patients and restored the normal activity of their serpina1 gene.
Dozens of serpina1 alleles are found in the human population. The most common is the allele designated M which encodes alpha-1-antitrypsin a protein of 394 amino acids. The most common defective allele is one designated Z. It is an autosomal recessive so homozygotes (ZZ) are a risk of all the complications of alpha-1-antitrypsin deficiency.
The protein produced by the M gene has a glutamic acid codon [GAA or GAG] at position 342. The mutant Z gene encodes a lysine [codons AAA or AAG] at that position. This alteration produces a misfolded, non-functional protein. The CRISPR construct snips out an A from the lysine codon replacing it with a G thus forming the M, healthy, alleles.
What makes this such an epoch-making event?
Up to now, the spectacular advances in gene therapy have involved
eitheradding healthy replacement genes. Follow this LINK for several examples.
or
breaking a harmful gene to silence it. Two examples: PCSK9 (above); and C1INH deficiency).
This new methodology restores the ability of the patient's own liver cells to secrete a fully functional alpha-1-antitrypsin protein. It also has the advantage over the other gene editing techniques (above) in only needing a single treatment for, one hopes, a lifetime of function. The effectiveness of most other gene editing techniques wanes over time
.| Welcome&Next Search |