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CRISPR-Cas9

Clustered regularly-interspaced short palindromic repeats (abbreviated as CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. CRISPR is being used as a tool that allows scientists to edit genomes with unprecedented precision, efficiency, and flexibility. CRISPR is far better than older techniques for gene splicing and editing (see video 1).

The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. CRISPR spacers recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. A set of genes was found to be associated with CRISPR repeats, and was named the cas, or CRISPR-associated, genes. The cas genes encode putative nuclease or helicase proteins, which are enzymes that can cut or unwind DNA. The Cas genes are always located near the CRISPR sequences. There are a number Cas enzymes, but the best known is called Cas9, which comes from Streptococcus pyogenes.

The CRISPR interference technique has enormous potential application, including altering the germline of humans, animals and other organisms, and modifying the genes of food crops. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. CRISPRs have been used in concert with specific endonuclease enzymes for genome editing and gene regulation in species throughout the tree of life. Ethical concerns have been expressed about this nascent biotechnology and the prospect of editing the human germline.

Video 1. Genome Engineering with CRISPR-Cas9, explained by Jennifer Doudna (UC Berkeley). Jennifer Doudna is credited for her biochemistry research to determine the mechanism of and to create the CRISPR/Cas9 system.

Mechanism
CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system. Cas9 is an enzyme (nuclease) that cuts DNA, and CRISPR is a collection of DNA sequences that tells Cas9 exactly where to cut. A guide RNA is required to feed Cas9 the right sequence, where to cut and paste bits of DNA sequence into the genome wherever you want. When utilized for genome editing, this system includes Cas9, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) along with an optional section of DNA repair template that is utilized in either Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). The crRNA contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9. The tracrRNA binds to crRNA and forms an active complex with Cas9.

CRISPR/Cas9 often employs a plasmid to transfect the target cells. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the cell's DNA. The crRNA must bind only where editing is desired. The repair template must also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence. Multiple crRNA's and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. The Cas9 protein with help of the crRNA finds the correct sequence in the host cell's DNA and creates a single or double strand break in the DNA. Properly spaced single strand breaks in the host DNA can trigger homology directed repair, which is less error prone than non-homologous end joining that typically follows a double strand break. Providing a section of DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9 induced DNA break. The goal is for the cell's HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells (see video 2).

Applications
Like RNAi, CRISPR interference (CRISPRi) turns off genes in a reversible fashion by targeting, but not cutting a site. The targeted site is methylated so the gene is epigenetically modified. This modification inhibits transcription. Cas9 is an effective way of targeting and silencing specific genes at the DNA level. Cas9 was used to carry synthetic transcription factors (protein fragments that turn on genes) that activated specific human genes. CRISPR simplifies the creation of animals for research that mimic disease or show what happens when a gene is knocked down or mutated. CRISPR may be used at the germline level to create animals where the gene is changed everywhere. CRISPR can also be utilized to create human cellular models of disease. For instance, CRISPR was applied to human pluripotent stem cells to introduce targeted mutations in genes relevant to two different kidney diseases, polycystic kidney disease and focal segmental glomerulosclerosis.

Video 2. What is CRISPR/Cas9? How does CRISPR system work? What is needed to perform genome engineering in your lab? This short video introduces the basics of this novel technology, as well as genome editing/knockout products offered by OriGene.

Human germline modification
In April 2015, scientists from China published a papr (Protein Cell. 2015 May; 6(5): 363–372) reporting results of an attempt to alter the DNA of non-viable human embryos using CRISPR to correct a mutation that causes beta thalassemia, a lethal heritable disorder. The experiments resulted in changing only some of the genes, and had off-target effects on other genes. The scientists who conducted the research stated that CRISPR is not ready for clinical application in reproductive medicine. This publication raised serious concern about editing genes in embyos (see Chinese Scientists Edit Genes of Human Embryos, Raising Concerns.

In December 2015, the International Summit on Human Gene Editing took place in Washington. Members of national scientific academies of America, Britain and China discussed the ethics of germline modification. In conclusion, they agreed to proceed further with basic and clinical research under appropriate legal and ethical guidelines. A specific distinction was made between clinical use in somatic cells, where the effects of edits are limited to a single individual, versus germline cells, where genome changes would be inherited by future generations. This could have unintended and far-reaching consequences for human evolution, genetically (e.g. gene/environment interactions) and culturally (e.g. Social Darwinism), hence altering of gametocytes and embryos to generate inheritable changes in humans was claimed irresponsible. In addition, they agreed to initiate an international forum where these concerns will be continuously addressed, and regulations in research harmonised across countries. In February 2016, British scientists were given permission by regulators to genetically modify human embryos by using CRISPR-Cas9 and related techniques.

On February 8th, 2015, U.S. director of national intelligence added in the annual worldwide threat assessment report of the U.S. intelligence community, gene editing to a list of threats posed by “weapons of mass destruction and proliferation.” It is gene editing’s relative ease of use that worries the U.S. intelligence community, according to the assessment. “Given the broad distribution, low cost, and accelerated pace of development of this dual-use technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications,” the report said. Although the report doesn’t mention CRISPR by name, Clapper clearly had the newest and the most versatile of the gene-editing systems in mind. The CRISPR technique’s low cost and relative ease of use—the basic ingredients can be bought online for $60—seems to have spooked intelligence agencies.