Please note: The following article has been included under other conventional therapies because a separate section is not yet available for experimental molecular biology methods outside of human medicine. The CRISPR/Cas method is a molecular biological method for the targeted cutting as well as modification of DNA (genome editing; gene scissors). In 1987, scientists discovered a previously unobserved adaptive immune system in E. coli. This is based on so-called CRPSPR sequences (clustered regularly interspaced short palindromic repeats) within the DNA. E. coli integrates the DNA of bacteriophages (groups of viruses that specialize in bacteria as host cells) the CRSPR sequence of its own DNA, thus transcribing a crRNA (rewriting DNA into RNA). The crRNA consists of both spacer and repeat sequences. Spacer sequences are the sequences “extracted” from the bacteria. The so-called trRNA (tracrRNA) binds to named repeat sequences. It recruits the CAS9 enzyme. A complex is now present – the crRNA:tracrRNA:Cas9 complex – which is capable of binding bacteriophage DNA complementary to the space sequences of the crRNA. As a so-called endonuclease (DNA-cutting enzyme, thus a restriction enzyme), CAS9 cuts the viral DNA in a double-stranded manner, which ultimately leads to replication inability (i.e. no further replication and consequently no further integration). For more than a decade, this procedure has been used with emphasis in genome editing research. Described “crRNA:tracrRNA:Cas9 complex” is universally applicable to plants and animals and allows removal (deletion) and ultimate silencing of genes. A use outside of research has been found for more than 5 years in agriculture and food crops for drought tolerance as well as immunization enhancement against viral pathogens. The procedure could possibly be used in human medicine later on. Since 2020, for the first time, there is a curative therapeutic approach for a disease with a congenital heart defect (vitium) in children. Vitium is part of the complex hereditary disease Noonan syndrome (autosomal recessive or autosomal dominant inheritance). After deciphering the causal variants of the LZTR1 gene, appropriate gene correction of the generated induced pluripotent cardiomyocytes (heart muscle cells) from stem cells of the twins was performed. The gene regulates essential signaling pathways for cell differentiation and growth.
Before this potential therapy in human medicine
Molecular genetic testing for inherited disorders in parents including comprehensive genetic counseling.
The procedure
The procedure is similar to that of the defense mechanism of E. coli described in the immune system. In this process, the spacer portion of crRNA can be modified to cut sequence-specific double-stranded complementary DNA, resulting in targeted deletions. The chemically modified trRNA:crRNA molecule is called guideRNA. This requires two different crRNA:tracrRNA:Cas9 complexes to attach to two sites on the DNA. After removal of the DNA fragment, enzyme-assisted linkage of the 2nd DNA fragments occurs by ligases. This differs from the mere cutting of a DNA sequence as in the bacterium. Over the years, essential modeling techniques have been added. These allow not only deletions within the DNA strand, but also the addition (insertions) of new DNA nucleotides. The most promising modification is prime editing. Here, a deletion and thus removal of a DNA fragment is followed by the insertion of a new DNA fragment. The so-called pegRNA (prime editing guide RNA) is available here as the transcript of the DNA to be inserted. With the aid of a reverse transcriptase, the pegRNA is transcribed into DNA and incorporated into the DNA, again using ligases. The CAS9 protein required for this process generates single-stranded instead of double-stranded cuts. This allows the new DNA fragment to be inserted with a precise fit into the cut DNA strand with its protruding ends. The new modification is fundamental for exchanging the “pathological” DNA sequence according to the “non-pathological” one in the future in the context of a hereditary disease.
After therapy
Once again, genome screening is performed to confirm the success of genome editing.
Possible complications
Due to possible base mismatches of guideRNA, off-target effects, i.e., binding at the undesired site, may occur. These can cause point mutations (base changes), insertions (incorporation of additional nucleotides or DNA sequences into a DNA sequence), deletions (loss of …), translocations (change in position of DNA), and inversions (presence of a DNA segment turned 180 degrees). The CAS9 enzyme does not cut at the desired location in every case. However, increases in specificity have already been realized through changes in protein design. Also, by linking CAS9 to an endonuclease Fokl, also derived from bacteria, specificity could be increased to 1: 10,000 (without other modifications, only specificity of up to 1: 2).
