History of CRISPR
CRISPR was first discovered in 1987 when Japanese researchers found unusual repeated sequences in the genome of E. coli bacteria. They could not determine the purpose of these repeats at the time. In 1993, researchers found similar repeats in many other bacteria and archaea. It was not until 2005 that CRISPR's adaptive immune function in prokaryotes was characterized. CRISPR sequences were found to incorporate small fragments of the genomes of invasive viruses or plasmids into their CRISPR loci as an adaptive immune response.
Cas proteins, such as Cas9, are encoded near the CRISPR loci and are involved in the destruction of foreign nucleic acids bearing complementary sequences to CRISPR spacers. This adaptive prokaryotic immune system provides resistance and immunological memory that is heritable. The potential biotechnology applications of manipulating this system for precise genome engineering were quickly realized.
Cas9-Based Genome Editing
In 2012, Jennifer Doudna and Emmanuelle Charpentier published the seminal research demonstrating that Streptococcus pyogenes Cas9 endonuclease could be easily programmed by a synthetic single guide RNA to induce double-stranded breaks at targeted genomic loci in human and bacterial cells. This breakthrough established the simplicity and wide applicability of using a Cas9-gRNA complex for genome editing. Later that year, Feng Zhang demonstrated the same capability of Cas9 from Streptococcus thermophilus.
Cas9 is now the central component of most CRISPR-based genome editing systems. The Cas9-gRNA complex scans DNA for complementary gRNA-specified target sequences and induces a double-stranded break at that site. Cells have mechanisms to repair double-stranded breaks, primarily through non-homologous end joining or homology-directed repair. Non-homologous end joining is error-prone and often results in insertions or deletions that can alter the target sequence. Homology-directed repair uses a repair template to precisely insert or modify the targeted DNA.
Disease Modeling and Therapeutic Correction
CRISPR technology enables new possibilities for modeling diseases and developing therapies. Disease mutations can be introduced into cells or animal models via Cas9 genome editing to study disease pathogenesis and evaluate treatment strategies. This provides advantages over current models. Concurrently, therapeutic genome editing aims to directly correct disease-causing mutations in vivo.
One application is modeling genetic diseases such as cystic fibrosis by introducing the common ΔF508 mutation into cell lines and animal models. Researchers can now better study the disease mechanism and test CFTR modulator drugs. Similarly, cancer cell lines can be engineered to investigate oncogenic mutations and evaluate therapies. Cas9 offers precise genome editing capability applicable to numerous genetic disorders involving single-nucleotide mutations, insertions, deletions, and more.
Beyond correction of Mendelian genetic disorders, CRISPR and CAS Gane -based cures targeting somatic or stem cells are being explored for conditions with diverse causes such as hematological malignancies, neurodegenerative diseases, and viral infections. Challenges remain around efficient delivery, specificity, and immune response to Cas9 and DNA repair machinery. However, clinical trials are ongoing to evaluate safety and efficacy. As the technology advances, CRISPR therapies could revolutionize treatment of currently incurable diseases.
Challenges and Safety Concerns
While CRISPR disease modeling and therapies hold enormous promise, challenges still need to be overcome before broad clinical applications. One concern is off-target editing at sites with imperfect matches to the gRNA which could potentially cause harmful mutations. Methods to enhance Cas9 specificity continue to improve, such as paired Cas9 nickases or fusions to tracking domains, but off-target effects remain a factor to thoroughly evaluate.
Delivery of the Cas9 components as well as DNA repair templates to target cells or tissues also requires optimization. Viral vectors achieve high transduction but carry safety risks. Non-viral delivery methods are being pursued but generally have lower efficacy. CRISPR effects may also vary between cell types and individuals depending on DNA accessibility, chromatin state, and DNA repair pathway efficiency. Unintended changes to gene expression profiles from Cas9 and repair machinery presence could theoretically alter cell behavior.
Ethical issues also warrant consideration as the technology develops. Genome editing capabilities could enable enhancement of human traits, raising questions about germline editing and eugenics. Regulatory frameworks are needed as clinical trials advance. Careful preclinical evaluation is important to fully address technical limitations before advancing to human applications. With responsible research, CRISPR has potential for monumental advances in biomedicine.
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