Introduction to CRISPR/CAS9

History

In the early 2000s, researchers developed Zinc Finger nucleases which are synthetic proteins whose DNA-binding domains enable them to cut DNA at specific locations.

Later, synthetic nucleases called TALENs provided an easier way to target specific DNA and were predicted to surpass zinc fingers in functionality.

Both Zinc Finger and TALENs depend on making custom proteins for each DNA target which is still a fairly cumbersome procedure.

In 2012, CRISPR/CAS9 developed which uses guide RNA. This system is efficient and can target more genes than earlier techniques.

What is CRISPR/CAS9

CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPR was first shown to work as a genome engineering and editing tool in human cell culture. It has since been used in a wide range of organisms including baker's yeast, zebra fish, nematode worms, plants, mice and several other organisms. Additionally, CRISPR has been modified to make programmable transcription factors that allow scientists to target and then activate or silence specific genes.

Libraries of tens of thousands of guide RNAs are now available.

The first evidence that CRISPR can reverse disease symptoms in living animals was demonstrated in March 2014, when MIT researchers cured mice of a rare liver disorder.

CAS9 = CRISPR Associated genes CAS9 is an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. We are able to use CAS9 to home in on a specific location that we would like to cut and edit DNA.

Functions

Editing

CRISPRs can add and delete base pairs at specifically targeted DNA loci. CRISPRs have been used to cut as many as five genes at once.

Reversible knockdown

CRISPR interference "CRISPRi" turns off genes in a reversible fashion by targeting but not cutting a site. In bacteria, the presence of CAS9 alone is enough to block transcription, but for mammalian applications, a section of protein is added. Its guide RNA targets regulatory DNA, called promoters that immediately precede the gene target.

Activation

CRISPR interference CAS9 has been used to carry synthetic transcription factors (protein fragments that turn on genes) that activate specific human genes. The technique achieves a strong effect by targeting multiple CRISPR constructs to slightly different locations on the gene's promoter. The genes include some tied to human diseases, including those involved in muscle differentiation, cancer, inflammation and producing fetal hemoglobin.

Applications

A featured company, "Editas Medicine" which is a well funded startup, aims to develop treatments that employ CRISPR/CAS9 to make edits to single base pairs and larger stretches of DNA. Inherited diseases such as cystic fibrosis and sickle-cell anemia are caused by single base pair mutations; CRISPR/CAS9 technology has the potential to correct these errors. Before gene editing can be used clinically, the company must be able to guarantee that only the targeted DNA region is affected and determine how to deliver the therapy to a patient’s cells.  Other pathologies potentially treatable by CRISPR include Huntington’s disease, aging, schizophrenia and autism, not to mention modifying DNA in living embryos. Improved targeting is required before CRISPR can be used in medical applications.

Legal

A provisional US patent application on the use of the CRISPR system for editing genes and regulating gene expression was filed by the inventors on May 12, 2012. Subsequent applications were combined and on March 6, 2014 the resulting patent application was published by the USPTO. The patent rights have been assigned by the inventors to the Regents of the University of California and to the University of Vienna.

Future

There have been over 130 papers published on CRISPR/CAS9 since the technique was first discovered. Three startups have been founded focusing on specific disease and conditions to cure.

Addgene Repository

Free software is available to design RNA to target any desired gene. The Addgene repository offers academics the DNA to make their own CRISPR system for $65. In 2013, Addgene distributed more than 10,000 CRISPR constructs. The facility has received CRISPR-enabling DNA sequences from 11 independent research teams.

Commongene.org

An organization that creates unity through genetics for the purpose of proliferating healthy life througout the universe.