The CRISPR-Cas9 System

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat, referring to the repeating DNA sequences found in the genomes of microorganisms. CRISPR technology allows scientists to make precise changes in genes by splicing and replacing these DNA sequences with new ones. Through these changes, the biology of the cell is altered and possibly affects the health of an organism. The possibilities are endless as this offers opportunities in curing deadly diseases, modifying genes, and changing humanity as we know it. Although bioengineering has been around since the 1960s, CRISPR is significant because of the comparative low costs and the ease of the procedure to

The author gives a brief history of past genome editing but thoroughly explains the history and mechanism of the CRISPR technology. She elaborates on how the technology has already been used to cure diseases and speculates on its future uses and regulation.

So, in one study, scientists engineered a modified CRISPR system to work with customized guide RNA (gRNA) in human cells to target genes linked to leukemia, to which they obtained high targeting rates. They proved that this process was not by random chance, but that it was caused by CRISPR components, that the process was sequence-specific and, when they introduced multiple gRNAs, it can affect multiplex editing of target loci. Their results created an RNA-guided editing tool for easy, efficient, and generalized human genome

The endonuclease Cas9 is the core multifunctional protein of the CRISPR-Cas9 system. Two nuclease domains: HNH (39) and RuvC-like (40), within the Streptococcus pyogenes Cas9 (SpCas9) protein conduct the function of cleaving DNA strands. Studies have confirmed that the tracrRNA: crRNA duplex structure will lead to the sequence-specific DNA cleavage (41). PAM short sequence facilitates crRNA to recognize the targeted DNA sequence (42). Cas9 protein cuts the specific DNA strand that is complementary to the 20bp crRNA sequence via HNH domain; on the other hand, the SpCas9 RuvC-like domain cleaves the opposite targeted DNA strand (42) (Fig. 4). Mutating either of these two nucleases domains of SpCas9 will introduce a single-stranded DNA nick, while mutating both HNH and RuvC-like domains will transform SpCas9 into a RNA-guided DNA binding protein (42). X-ray crystallography and has confirm that endonuclease Cas9 undergoes a series of conformational rearrangements when it binds to the guide RNA and target DNA (55).

Genes are the foundation of life. Biologists throughout history have studied genes with the goal of knowing how they work, what functions specific genes have, and what they can do with this knowledge to further science and society. Techniques involving genes have grown to the level where we can target specific genes to edit them and even alter their function. Some earlier techniques of this include meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, but each of these techniques has its disadvantages (Sander and Joung, 2014). Recently in the world of genetics, a new and exciting method to target specific genes easily and with high precision has emerged. Clustered regularly interspaced short palindromic

The CRISPR/CAS9 is a genome engineer technique that allows one to make somewhat precise cuts of DNA within three base pairs that then allows either the insertion or deletion of DNA. CRISPR stands for “Clustered regularly interspaced short palindromic repeats”, CAS9 is but one variation of the CAS proteins, and Cas9 stands for “CRISPR associated protein 9”. Cas9 is a protein that induces site-directed double strand breaks in DNA. The CRISPR/CAS9 uses a guide RNA to recognize complementary 20 nucleotide base pairs to the spacer region of the guide RNA. If the DNA is complementary to the guide-RNA, the Cas9 cleaves the DNA, allowing a portion of the genome to be deleted hence it can create a knock out gene. CRISPR/CAS 3, 9 and 10 were discovered in archaea and bacteria and they were felt to be the adaptive immune system of bacteria and archaea against bacterial phages and F-plasmids and viruses. It is considered an adaptive immune system since they discovered that bacteria with resistance to multiple phages had multiple inserts that offered immunity and in particular it was this Cas 3,9 and 10 protein that offered immunity by cleaving out the invading DNA. The technique is much more complicated than Jennifer Doundna explained in her TedTalk. However, it much less complicated than say using Zinc fingers or TALENS for

The CRISPR/Cas9 system requires a proto-spacer adjacent motif (PAM) sequence. The PAM is necessary for cleavage of target DNA by Cas9, allowing differentiation between invading viral genome (in this case HIV) and the CRISPR locus in the host genome. One limitation of the CRISPR/Cas9 system to disrupt HIV could be due to low altering specificity, as the target loci is very small, so significant off target cleavage can occur (Wang et al.). CRISPR-Cas9 may create non-specific mutations in areas of the genome with similar homology, outside of the HIV provirus locus, which may cause cellular transformation (Larson et al., Tu et al.). Selection of Cas9 homologs with a longer PAM may decrease the probability of non-specific gene target and Cas9 homologs

Regardless of those burning problems, CRISPR/Cas9 genome-editing technique presents staggering opportunities for addressing a number of illnesses beyond the reach of previous treatment modalities. Taking into account the accelerating rate of technological progress, as well as a wide range of research and clinical applications, the road ahead of us will certainly be a thrilling

This article discusses scientists from Oregon Health and Science University who successfully edited the DNA of human embryos to eliminate a mutant gene of a heritable heart condition, using gene editing technology known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). They focused on an inherited heart disease called hypertrophic cardiomyopathy, which is the potential thickening of the heart muscle. The procedure consisted of using molecular scissors to cut out the mutation in the sperm right before it fertilized the egg. The embryo then repaired the cut in the sperm’s genetic material by copying the egg’s healthy gene, leaving the embryo free of a genetic disease that would otherwise be passed down by the father. It

Hsu, P., Lander, E., & Zhang, F. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262-1278. http://dx.doi.org/10.1016/j.cell.2014.05.010

The problems that may arise by using CRISPR-Cas technology for germline cell editing is the possibility of mosaicism that may occur in the edited genes. Mosaicism refers to the fact that the cells of the embryo that has been edited may result in altered cells and diseased cells, therefore the embryo still has the potential for disease. Another potential obstacle that researchers may have to face while using this genome editing technology is if the components that have been edited will possibly bind to genomic regions that are targeted by the RNA guide because the sequence is extremely

The CRISPR-Cas9 complex is derived from the immune system of bacterial cells and also contains repurposed exogenous RNA’s responsible for editing the human genome.1 To edit a gene within the genome, researchers add a CRISPR RNA (crRNA), which is complementary to the DNA code of interest and is responsible for binding. The crRNA is engineered to be extremely specific to the code of interest. A trans-activating CRISPR RNA (tracrRNA) is also necessary for guiding all the pieces of the complex together to carry out the function of editing. The repurposed CRISPR-Cas9 complex contains two nucleases, RuvC and HNH, which perform noncomplementary strand cleavage and complementary

The question that should be asked now is how the CRSIPR sequences simply defend the micro-organism from invading viruses. In figure one; below there is a sequence of black diamonds (which represent DNA) and in between them are spacers. This is what is known as a CRISPR sequence. The spacers are vital for the immunity of the micro-organism or bacteria. They contain the genetic information of the previous viruses that have attacked this micro-organism. Furthermore, when a new virus attacks the micro-organism, a new spacer is added to the CRISPR sequence. This allows the immune system to recognise and silence exogenous genetic material. The process of this happens as the DNA of the virus is cut into short segments by the Cas (CRISPR associated)

Landers would like to ask the question if screening system (CRISPR/CAS9) would be a good tool to use in analyzing genetics in mammalian cells (5). Lander’s background researched in the area of Mammalian Genomes led him to find a process noted in this paper (3). According to the ‘Abstract’ section of this paper, Landers hypothesized that using this new process would help rapid generation of certain cells (5). Landers tested his hypothesis using the experiment with mice cells

Genome editing is a huge leap forward in science and medicine. Because of recent advances in technology, the study of genes and induced ‘point’ mutations have led to the discovery and advancement of methods previously used in order to mutate genes. The development of Clusters of Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR associated system 9 protein (Cas9) technology is a hugely significant leap forward as this is a tool that could potentially be used for the research into and hopefully the treatment of a range of medical conditions that are genetically related. Cystic fibrosis (Schwank, G. et al, 2013), haemophilia and sickle cell disease are an example of some of the conditions that have the