CRISPR/Cas9

CRISPR's (Clustered Regularly Interspaced Short Palindromic Repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. It is pronounced "crisper".  See Restriction enzymes for critical difference

CRISPR can also be defined as an RNA guided endoculease system, evolved in bacteria as a defensive approach to protect the cells from viruses (Bacteriophages) attack.

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. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea.

The CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be relatively cheaply cut at any desired location. CRISPR has a number of potential applications including altering the genetic blueprint of humans and livestock, and manipulating the genes of food crops, but the application of this method is accompanied by ethical concerns,  see article in thoughts on being used for the first time in humans.

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CRISPR Cas9 gene editing tools have been adapted from a prokaryotic immune system to provide sequence specfic genome targeting of any species.  The nuclease introduces double stranded breaks.  The break is repaired by one of two mechanisms: 1. Non-homologous end joining (NHEJ) which creates random insertions or deletions (indels) at the targeted site or 2. Homologous recombination which creates precise changes based on template DNA.    HDR

Genome Editing

CRISPRs can add and delete base pairs at specifically targeted DNA loci and have been used to cut as many as five genes at once. Or up to 62 genes at once—Pig cells have been engineered to inactivate all 62 Porcine Endogenous Retrovirus in the pig genome using CRISPR Cas9 genome editing technology, and eliminated infection from the pig to human cells in culture. CRISPR's low cost compared to alternatives is widely seen as revolutionary.

Selective engineered redirection of the CRISPR/Cas system was first demonstrated in 2012 in the following applications:

---Immunization of industrially important bacteria, including some used in food production and large-scale fermentation

---Cellular or organism RNA-guided genome engineering. Proof of concept studies demonstrated examples both in vitro and in vivo

---Bacterial strain discrimination by comparison of spacer sequences

Cas9 Restriction Enzyme.   see wikipedia opposite--see its own entry also

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to memorize and later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking whether it is complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes.

Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and TALEN proteins, Cas9 is becoming a prominent tool in the field of genome editing.

Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and Zinc-fingers), engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. While native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide - the CRISPR RNA (crRNA), and the trans-activating RNA (tracrRNA)., Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA. Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. In 2015, scientists used Cas9 to modify the genome of human embryos for the first time.

Protospacer adjacent motif (PAM) is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component (not found in bacteria) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.

What is a PAM sequence and where is it located?

CRISPR/Cas9 mechanisms recognize DNA targets that are complementary to a short CRISPR sgRNA sequence. The part of the sgRNA sequence that is complementary to the target sequence is known as a protospacer. In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.

Recognition of the PAM by the Cas9 nuclease is thought to destabilize the adjacent sequence, allowing interrogation of the sequence by the sgRNA, and resulting in RNA-DNA pairing when a matching sequence is present [1, 2]. Cas9 nucleases with alternative PAMs have also been characterized and successfully used for genome editing [3]. It is important to note that the PAM is not present in the sgRNA sequence but needs to be immediately downstream of the target site in the genomic DNA. Usually NGG where N is any nucleotide.

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Smallest CRISPR So Far Comes from Food Poisoning Bacteria--  

The Cas9 enzyme is used by several bacteria as an immunity weapon—it is needed to cut viral DNA that could damage the bacteria. The most common version of the CRISPR/Cas9 technique uses Cas9 derived from the bacterium Streptococcus pyogenes. However, this protein is made of 1368 amino acids, and it is too large to be delivered and packaged in AAV. Even if scientists split it up into two parts, each packaged in a different virus, other issues arise. A double amount of viruses need to be delivered, and the split Cas9 is less active than the intact SpCas9. Over the years researchers have discovered smaller versions of the Cas9 enzyme, such as the one derived from Staphylococcus aureus (1053 amino acids). However, while this version can just fit inside the AAV, it does not leave enough space for other essential proteins.

In the current study, the research team found that C. jejuni Cas9 is both efficient and small. At 984 amino acids, it can be packed into AAV together with multiple guide RNAs, as well as a fluorescent reporter protein.

“We present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo,” the authors wrote. “After determining protospacer-adjacent motif (PAM) sequences and optimizing single guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome.”

To use a bacterial protein for gene editing, the investigators had to optimize some aspects of the technique. They designed a short DNA sequence immediately following the sequence targeted by the Cas9, called the protospacer adjacent motif (PAM). Each different Cas9 needs a specific PAM sequence, otherwise, it will not be able to bind to and cleave the target DNA sequence. Additionally, they had to modify the length of the guide RNA.

Supplanted by a Single Nucleotide system in Mice, see   "Single Nucleotide Editing..." Comparison of the most used CRISPR system (top) vs. the newly developed editing tool (bottom). In some cases, the difference of just one nucleotide can bring serious disease consequences. Scientists hope to cure such diseases by substituting the incorrect nucleotide with the correct one. [IBS]

January 19, 2018

CRISPR Activation of Single Genes Turns Skin Cells to Stem Cells

A new way of inducing pluripotency has been demonstrated by scientists based at the Gladstone Institutes. Rather than fuss with the usual transcription factors or drug-like chemicals, the Gladstone scientists, led by Sheng Ding, Ph.D., used CRISPR activation (CRISPRa). This technique, which incorporates a modified form of CRISPR gene-editing systems Cas9 enzyme, allowed Dr. Ding and colleagues to target and epigenetically remodel specific genomic locations: the Oct4 and Sox2 genes.

Remodeling either gene, the scientists discovered, turned skin cells from mice into stem cells. Detailed findings appeared January 19 in the journal Cell Stem Cell, in an article entitled “CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency.”

“...single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network,” wrote the article’s authors. “...targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming.”

Feb1 2018--lovely 3D video of CRISPR/Cas9 working, see Tech Crunch opposite. Watch what it actually......  Enlarge poster below--good definition  see also CRISPR gene editing explained Mar 2019 opposite

See 2020 article for Mar 2020 update--  <img src="/e9a3a7e1-6844-5abc-a515-7e7444eae76c.png"/>  target--insert--fix--all together!!

CRISPR/Cas9

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