CRISPR technology has revolutionized laboratory cell modification. However, providing this tool to diseased cells in vivo still presents its own set of obstacles.
Scientists derived this technology from natural defense mechanisms found in bacteria. Such microbes use captured pieces of invader DNA as guidance for molecular machinery to damage and slice up their DNA genomes.
What is CRISPR?
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and refers to DNA sequences found in bacteria that contain repeated segments of genetic code interrupted by “spacer” sequences – these “spacer” sequences serve as an immune memory against future attacks on their target bacterium, creating an adaptive immunity against invading viruses known as phages. Scientists first observed CRISPR systems in archaea before their discovery in bacteria, functioning as part of an adaptive defense mechanism against attacks by invading viruses known as phages.
Scientists have used CRISPR technology in the laboratory to modify genes of organisms as diverse as fruit flies, fish, mice, plants and human cells. By easily altering gene activity across species such as these one, scientists are able to study various aspects of biology such as how disease-causing genes operate – this same technology could even be adapted to treat diseases by inserting functional genes directly into cells.
CRISPR uses an enzyme known as Cas9 that acts like molecular scissors. Cas9 can cut DNA at specific sequences when guided by customizable guides — these could include DNA, RNA or any chemical compound — while scientists can customize this process using variants of Cas9 as well as guide RNAs that target certain chromosomes or subsets thereof.
In 2017, UC Berkeley researchers pioneered an effective new way of using Cas9, called CRISPR-Cas9. By creating a custom Cas9 variant that could target specific chromosomes within cells, scientists could modify specific genes while leaving others intact – this technique has become the standard method for genome engineering.
CRISPR can be difficult to deliver into cells. Some scientists have tried slipping it into lab dishes with cells in them, but it can be challenging to target specific types of cells – for example if trying to edit liver cells with CRISPR may lead it instead into muscle cells instead. Other researchers are working on developing ways for CRISPR delivery directly into living patients.
CRISPR-Cas9 (Cas9)
CRISPR-Cas9 is an impressive genome engineering tool, enabling scientists to make targeted edits to living cells’ DNA. This two-part system comprises Cas9 (a nuclease that cuts DNA) and its target guide RNA (gRNA), both designed to direct Cas9 towards specific target sequences within their genomes. CRISPR-Cas9’s precision and ease of use make it one of the leading gene editing technologies – used widely both in research and medicine including correcting or inserting new genes into cells.
The technology relies on an existing natural defense mechanism in bacteria to combat viruses; to do this they capture small pieces of their genetic material which later help an enzyme called Cas find and dismantle viruses’ genetic material.
Scientists have now used CRISPR-Cas9 technology in various organisms, from human cells and mice to bacteria and algae. By targeting specific genes, researchers can create “knockout” cells or animals without certain functions for that gene, as well as use this system to gain more insight into how genes function in a cell.
For researchers to use CRISPR-Cas9 effectively, they first must design a guide RNA (gRNA). A gRNA contains both the guide sequence that matches DNA targets in cells as well as complementary RNA that allows it to bind tightly with it and initiate cutting. After designing their gRNA, plasmid or viral vector delivery of it may also be needed to reach cells.
Once bound to its target DNA strand, Cas9 nuclease cuts it with precision using its cutting action to generate double-stranded breaks that may then be repaired using your cell’s own repair mechanisms or used as a template for inserting new genetic sequences.
CRISPR technology can often create new genetic sequences that encode therapeutic proteins for treating diseases. Already it has been used to treat rare genetic disorders, with other biotech and pharmaceutical firms working on CRISPR-based therapies for other conditions as well. Human clinical trials using CRISPR treatments are underway. Scientists are taking careful steps in order to assess its strengths and weaknesses as well as set best practices while discussing any social or ethical implications related to editing human genes.
CRISPR-Guide RNA (gRNA)
CRISPR is an impressive technology that enables scientists to manipulate the DNA of almost any living creature – from bacteria to humans. It offers greater ease, affordability, and precision than previous gene-editing techniques; with applications as diverse as curing genetic disease and creating drought-resistant crops.
Basic genome editing systems comprise two essential parts: Cas nuclease, which binds and cuts DNA; and guide RNA sequence (gRNA), which directs Cas nuclease to its target DNA strand. When combined, these elements form a ribonucleoprotein complex (RNP), which can be delivered into cells for genome editing. Multiple different gRNA sequences may be delivered simultaneously into one cell for genome editing; various tools exist to assist target site selection and gRNA design.
To target specific sequences of DNA, researchers need a gRNA with an attached Protospacer Adjacent Motif (PAM), such as 5’NGG3′. Researchers can use SnapGene Primer or similar tools to find suitable PAM sequences; the tool will highlight any matching nucleotides as well as sites likely to be cut by Cas9.
Once a PAM-matched target has been identified, it is necessary to create a gRNA sequence that will efficiently direct Cas nuclease towards it. When doing so, it is crucial that one chooses a sequence which complements target DNA with minimal off-target effects; additionally it’s also important to consider Cas9 protein’s cleavage efficiency in different systems or cell types.
As CRISPR technology advanced, new variations were designed to increase its specificity, efficiency and granularity. Such techniques include altering Cas proteins; using different guide RNA; and adding additional components.
CRISPR holds immense potential, but has yet to be fully exploited. Many scientists are proceeding cautiously as they assess its strengths and limitations, set best practices, and discuss any social or ethical implications related to human use of this tool. It is anticipated that CRISPR will become standard practice for genetic research soon despite these challenges.
CRISPR-Sequences
CRISPR is a cutting-edge technique used by scientists to edit the genetic makeup of living cells with precision. CRISPR works by cutting into DNA strands, allowing researchers to remove or insert specific sequences. CRISPR can be used for correcting defective genes that lead to diseases like cystic fibrosis, cataracts and Fanconi anemia – or just simply editing out unwanted sequences in DNA for general purposes such as correcting defects associated with cancerous growths such as cystic fibrosis or cataracts or Fanconi anemia.
Technology has driven research in areas as varied as synthetic biology, human gene therapy and disease modeling. Furthermore, this tool has expedited drug development as well as agricultural biotechnology, neuroscience and food science applications.
Scientists first observed CRISPR-Cas9 in nature when they discovered it being used by bacteria and archaeal microorganisms to ward off attacks by viruses. These microorganisms use CRISPR-Cas9 to cut up and store bits of virus DNA into their own genome for future reference; later this memory can help quickly destroy any repeat infections of that particular virus.
In 2012, American scientist Jennifer Doudna and French scientist Emmanuelle Charpentier co-published a paper in Science outlining their method to use Cas9 enzyme to edit DNA. Their technique utilized guide RNA for directed Cas9 digestion; their successful execution demonstrated this potential tool for gene editing.
Researchers have since perfected and extended its capabilities. By January 2013, scientists in Doudna’s lab and Feng Zhang’s, independently working, had found a way to engineer CRISPR-Cas9 to cut any DNA sequence found within mammalian cells.
CRISPR is so user-friendly that laboratories worldwide have quickly begun exploring it, finding many potential uses such as correcting defective genes in patients suffering from diseases like sickle cell anemia and beta-thalassemia that require frequent blood transfusions.
CRISPR technology does not come without its challenges, however. A major complication with CRISPR editing is off-target editing which may cut up and alter sections of DNA outside its target gene, potentially leading to mutations that lead to cancerous or otherwise damaged cells. Researchers are devising solutions such as sequencing cells after CRISPR modification for quality assurance purposes to make sure changes made are safe.
