CRISPR-Cas9

CRISPR-Cas9 is a genome editing system. The system originates in bacteria providing immunity to viruses and has been adapted for use as a genome-editing tool capable of knocking out genes and rewriting genetic sequences in animals, plants, and fungi. Outside of genome editing, modifications to the CRISPR-Cas9 system make it useful for gene regulation, genome imaging, and studying protein-genome interactions.

Cas9 is the nuclease enzyme that does the cutting in the Type II CRISPR systems used by Streptococcus thermophilis. The function of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) sequences that are part of a bacterial immune system was discovered in the yogurt bacteria Streptococcus thermophilus. Phillipe Horvath and Rodolphe Barrangou of Danisco (later DuPont) made that discovery and reported in 2007 in Science that the bacteria incorporate sequences from phage viruses they have been exposed to as spacers in the CRISPR region, which give the bacteria resistance to those phage viruses. DuPont has patented a technique of exposing bacteria to different phage viruses and uses CRISPR sequences to tell them which ones have acquired resistance, something that helps them avoid phage viruses spoiling their yogurt.

Horvath and Barrangou teamed up with biochemist Virginjus Siksnys at Vilnius University, Lithuania, and in 2012 they published how CRISPR works with Cas9. Around the same time, UC Berkeley’s Jennifer Doudna and Emmanuelle Charpentier (now at the Max Planck Institute for Infection Biology, Berlin) also described in Science how Cas9 works in the CRISPR system. Because they also engineered a simpler version of CRISPR that could likely work in other organisms including human cells, this transformed this bacterial immune function into a usable biotechnology tool.

There is a patent dispute over the invention of CRISPR-Cas9 technology specifically for use in human cells, between Doudna’s research team and Feng Zhang’s group at the Broad Institute (MIT and Harvard). Zhang’s research group and George Church’s lab at Harvard Medical School each published Science papers in 2013, showing they had modified CRISPR-Cas9 to edit the genome in human and mouse cells. The Broad Institute’s US patent, the first of several for mammalian use of CRISPR, is under appeal. Citing lack of novelty, the European patent office has revoked the first patent obtained by the Broad Institute and has granted patents to the University of California and University of Vienna. The first is for using the CRISPR-Cas9 system across prokaryotic and eukaryotic systems and the second is for a modified form of CRISPR-Cas9 to regulate gene expression.

Targeting of Cas9 to cleave DNA in bacterial immune function uses two RNAs that form a duplex, the crRNA that recognizes the invading DNA and the tracrRNA which hybridizes with the crRNA. Doudna’s group engineered the system to use a single guide RNA. The CRISPR-Cas9 genome engineering system uses a single protein, Cas9, and single guide-RNA complex (Cas9-sgRNA), and it is the most commonly used CRISPR system for gene editing. The CRISPR-Cas9 user-designed guide RNA binds DNA that contain the complementary sequence. The presence of a nearby (protospacer-adjacent motif) PAM sequence is required for cleavage in the target region. For endogenous CRISPR systems in bacteria, the absence of PAM sequences in the bacteria’s own genome prevents self-cleavage. In the human genome, the short PAM sequence is present at a frequency of 5.21%.

After CRISPR-Cas9 cleaves the DNA, the double-stranded break triggers repair by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ can knock out gene function due to random insertions or deletions occuring at the site that disrupt the reading frame or change the protein coded for by the sequence. Researchers take advantage of the HDR mechanism by supplying a user-generated DNA template that is used to correct the break. In this way, a gene mutation can be cut out and replaced with a corrected version of the gene. Scientists use CRISPR-Cas9 to alter genes in model organisms and cell lines to learn the function of those genes. CRISPR-Cas9 is also being used to develop therapies to treat or cure genetic diseases.

A modified form of CRISPR-Cas9 in which dCas9 cannot cut DNA maintains the ability to target DNA. When fused with transcriptional activators and repressors, it can turn gene expression up or down. Similarly, Epigenomic CRISPR-Cas9 systems are fused to proteins that recruit epigenetic modifiers to a target genomic region, resulting in changes in gene expression.

There are clinical trials at Hangzhou Cancer Hospital, China, and in the US by University of Pennsylvania researchers where the human immune cells, T-cells are removed from patients, modified by CRISPR-Cas9 in a way that enhances their ability to fight cancer, and put back into the patients.

In collaboration, CRISPR Therapeutics and Vertex Pharmaceuticals are using CRISPR-Cas9 outside the body to correct a genetic mutation in blood cells of patients with beta-thalassemia and sickle cell disease. Clinical trials for these diseases using CRISPR have been put on hold in the US to resolve questions about safety.

Editas Medicine is developing a CRISPR-Cas9 therapy for Leber's hereditary amaurosis. Autosomal recessive and autosomal dominant Retinitis Pigmentosa, choroidal neovascularization, and age-related Macular Degeneration are forms of blindness that are being tested with CRISPR-Cas9 preclinically in animal models.

CRISPR can fix the cystic fibrosis (CF) mutation in lung cells, intestinal cells, and iPS cells derived from patients Editas Medicine and CRISPR Therapeutics are working toward a therapy to use on CF patients.

Research in mice demonstrates CRISPR-Cas9 can fix Duchenne muscular dystropy (DMD) mutations. However, since there are many mutations that cause the disease in humans and one in three are new mutations, it is a challenge to design a gene-editing fix that works for more than just one mutation. Eric Olsen and his team at the University of Texas Southwestern Medical Center have developed a CRISPR-Cas9 gene-editing technique that targets 3000 types of DMD mutations. Their system uses 12 guide RNAs that target mutation hotspots to restore heart muscle function in human heart tissue derived from patients. Their system causes changes in splice sites so most commonly mutated sections of the RNA coding for the protein are skipped, a strategy called exon-skipping or myoediting. The resulting protein, while still incomplete, is still able to function well enough.