Bacterial CRISPR-Related Protein-9 Nuclease From Streptucoccus Pyogenes

Once the complex was bound to the DNA, a cut would be made to eliminate and destroy the invaders. 83% of archaeal genomes and 45% of bacterial genomes (Shabbir, M. et al, 2016) were shown to be able to successfully utilize the CRISPR Cas9 system. These are very promising statistics, so it is no wonder that there has been such an advancement in the past few years to bring this technology to eukaryotic cells, mammalian cells and eventually human cells.

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.

More recently, Kang et al have employed a different approach using a non-viral delivery method for CRISPR-Cas, known as the

CRISPR is versatile in that any target sequence can be modified by simply altering the gRNA sequence. In addition, multiple genes can be edited at the same time with great specificity (Cong 88-89). The convenience and accessibility of CRISPR resourced have also allowed thousands of laboratories worldwide to study CRISPR in different ways, which has broadened the horizons of its biomedical and clinical implications (Collins et al 259). Overall, the ease and simplicity of CRISPR technology has allowed for a rapid increase in the understanding of genome editing, which will allow CRISPR to revolutionize how certain conditions will be treated.

CRISPR-Cas9 is a gene editing tool that cuts out, replaces, and alters unwanted pieces of DNA. This process starts by scientists creating a synthetic RNA guide with a specific sequence. Upon entering the cell, the RNA guide meets up with the enzyme Cas9. The RNA then leads the Cas9 to a predetermined section of DNA. The Cas9 unzips the DNA which allows the RNA

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

Clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) are an immune response evolved by bacteria and archea as an adaptive defense mechanism to invading DNA. (4) The CRISPR Cas9 system relies on the uptake of invading DNA fragments that are then inserted into CRISPR loci. (4) In the CRISPR loci, repeats are separated by nucleotide spacers which match and or composed of invading DNA.(4) New spacer DNA is incorporated by Cas1 and Cas2.(4) The CRISPR spacer loci then transcribe into short CRISPR RNAs (crRNA) which anneal to foreign nucleic acids in conjunction with complementary binding trans-activating cr RNA(tracrRNA) to form a duplex which is then cleaved to provide a guiding RNA cr/tracr RNA hybrid.(4) the RNA hybrid acts as a guiding mechanism for Cas9 by complementary binding to the invading nucleotides.(4) Cas9 is an endonuclease that can cause a double stranded cleave in DNA(4) Cas9 guided with sgRNA then cleaves the foreign DNA resulting in double stranded breaks effectively disrupting and thereby removing a gene.(1)(2)(3)(4) After a ds break occurs cellular machinery attempts to fix the break with non homologous end joining in which cellular systems effectively sutures the broken ends of the DNA by recombining the remaining ends of DNA to once again produce a continuous strand.(4) This

(2012) introduced a new genetic technique that was derived from the defense mechanisms of bacteria. Some bacteria use a CRISPR-Cas system to defend against foreign viral and plasmid genetic material. Once foreign targets enter the system, the bacteria will integrate its CRISPR array to parts of the nucleotide sequences on the invading sequence. The bacteria will then produce a precursor CRISPR-RNA that complements the invading sequence, and is used to find all foreign sequences that match it. These precursor RNAs will work with Cas proteins to cleave the foreign sequence, thus effectively silencing it. There are multiple types of CRISPR-Cas systems that bacteria use. Type 2 systems, paired with Cas-9, use another RNA sequence, tracrRNA (trRNA), as a complement to precursor CRISPR-RNA. These systems used both trRNA and precursor CRISPR-RNA to induce a double stranded cleave. After this discovery, a Cas9 protein was purified and tested to see if it would be able to cleave DNA. It was discovered that if both a trRNA and a precursor CRISPR RNA were present with complementary sequences to a sequence in a DNA strand, the result would be a double strand cleave in the DNA. Cas9 also contains two domains, each of which only cleave either the complementary or the non-complementary strand of the target DNA. After looking at both the trRNA and the precursor CRISPR RNA, researchers theorized that they could engineer a chimera RNA that combined certain sequences of both

CRISPR Cas9 is a gene editing tool that can be used to edit, delete, and change parts of the genome. What makes CRISPR Cas9 different from other gene editing techniques such as Zinc Finger Nuclease (ZFN) or Transcription Activator-Like Effector Nucleases (TALEN) is its targeting efficiency ability. For example, ZFNs and TALENs could only reach targeting efficiencies from 1% to 50% in human cells. On the other hand, CRISPR Cas9 had a much higher rate at greater than 70% in Zebrafish and plants. (Reis, 2014). CRISPR Cas9 is still being heavily researched by scientists today, because although many advancements have been made since its discovery in 1993, it is far from ready to be used commonly. Successful studies and experiments with this gene

The CRISPR system uses two types of molecules, one of which is enzymes. One of the enzymes that can make up CRISPR is called Cas9. Cas9 cuts DNA at a specified location in the genome, determined by the guide RNA. This allows changes to be made in the genome by allowing DNA to be added or removed from

Phages are the most common organisms on Earth (3,7): they infect bacteria by injecting single-stranded DNA into their host cell, thereby altering the genetic code of the prokaryote. Once transcribed and translated, the injected DNA changes the function of the cell, often killing the host cell in the process (3). Over eons, archaea and some bacteria have adapted to such attacks with CRISPR-Cas systems, also known as clustered regularly-interspaced short palindromic repeats. The bacteria incorporate invading viral DNA into their genetic material in between redundant palindromic sequences. The viral DNA is then transcribed into pre-crRNA, where it combines with a Cascade complex. Cascade proteins come from CRISPR-associated (Cas) genes. Such genes code for small enzymes responsible for cleaving. They often join together into larger complexes known as Cascade, or CRISPR-associated complex for antiviral defense (2). The cas proteins use the RNA template as a guide and cut any other genetic material that matches that of the viral-template RNA, preventing the corrupt viral DNA from being transcribed into mRNA. As the number of spacers in the bacterium’s DNA increases, the level of phage resistance increases as well (1). Insofar, understanding of CRISPR and Cas genes is very limited. It is very much a twenty-first century discovery.

First discovered in 1987, the CRISPR-Cas system is a prokaryotic adaptive immunity defense system that confers resistance against invading nucleic acids, either viral or plasmid. In other words, if an intracellular parasite (such as a phage or a plasmid) invades the bacterial or achaeal host cell, the host cell is capable of storing short fragments of the foreign nucleic acid originating from the parasite within its own genome (Wiedenheft et al. 2012). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR associated (Cas) genes are found in approximately 40% of bacterial genomes and in nearly 90% of sequenced archaeal genomes (Zhang and al. 2014).

Even though CRISPR/Cas9 was just discovered around 4 years ago, the CRISPR/Cas system itself was first observed in bacteria (E.coli). It was what CRISPR was doing in the bacteria that makes it so unique. CRISPR stands for, “clustered regularly interspaced short palindromic repeats”. This means that there are going to be short sequences of DNA bases that will read the same forward and backwards. In-between these short sequences will be spacer DNA. Unlike the palindromic sequences, the spacer DNA will have sequences

The CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR associated protein) system is a defense mechanism found in bacteria and archaea to target and inactivate foreign DNA materials such as viral DNA. The foreign DNA is integrated into the CRISPR array. The inserted DNA, known as a spacer, is transcribed from CRISPR loci and translated into short CRISPR RNAs (crRNAs). The crRNA, also known as a guide RNA (gRNA), forms a complex with Cas9. This RNA-Cas9 complex scans for invading DNA materials complementary to the gRNA and cleaves it by Cas9, a protein encoded by CRISPR loci. Cas9 consists of an RNA binding domain, nuclease lobe for DNA cleavage, and protospacer adjacent motif (PAM) interacting sites recognizing the