Page tree
Skip to end of metadata
Go to start of metadata

A visualisation of CRISPR systems in action in bacteria and human cells © 2014 McGovern Institute for Brain Research at MIT
Occurring in the nucleus of a cell, CRISPR events are a complex ballet of DNA, RNA and protein, and happen stochastically, much like chemical reactions. Unlike chemistry, or the vast majority of enzyme-substrate reactions in biology, however, the key enzyme in the reaction, Cas9, which cuts DNA, is programmable, and can be guided to work specifically on almost any DNA target.

Many Elements Come Together to Cut DNA

The point of interest in a CRISPR event is the Cas9-sgRNA-DNA complex, or ribonucleoprotein. It is made up of the nuclease enzyme Cas9, which is bound to a guide RNA molecule (gRNA). The guide RNA has a specific sequence that directs the Cas9 nuclease to a corresponding DNA target in the genome. However, the Cas9-gRNA can only bind to a point on the genome that has a protospacer-adjacent motif (PAM) site, which is simply a specific sequence, such as "NGG". 

When the Cas9-gRNA complex finds a PAM site on one of the strands of DNA known as the non-target strand, the enzyme unwinds the double-helix structure of the genomic DNA. Following this, the Cas9 enzyme attempts to intertwine the gRNA with the other strand, known as the target stand. If there is a match between the gRNA and the genomic target strand, then the enzyme activates two separate nuclease domains, which cut both the non-target and the target stand, resulting in a double strand break (DBS). 

Broken DNA is Repaired

When the bacterial immune system targets invading viral or foreign DNA, the double strand break to the pathogenic DNA usually protects the bacteria from any harm - once broken, the invading DNA is rendered useless. When the double strand break is introduced to a segment of a cell's DNA in genome editing, that cell quickly recognises that a segment of its genome has been cut, and will attempt to initiate a repair.

Organisms vary in their preferred repair pathways, but most are able to use the Non-Homologous End Joining (NHEJ) pathway, which reliably repairs the double strand break by effectively "making up" a repair. The creation of a new section of DNA invariably sees a large number of errors being introduced at that site, and will typically disrupts the correct function of the current gene.

Alternatively, where a "donor" strand of DNA, that is sufficiently similar to the original section of DNA, is available, the donor can be integrated via the Homology-Directed-Repair (HDR) pathway, though this is not a reliable event and is very rare in CRISPR events other than in genome editing. 

The different CRISPR repair mechanisms and their use in genome editing is covered here:

How CRISPR Immunity Systems Differ from CRISPR Genome Editing Systems 

There are three primary types of CRISPR system found in bacteria in archaea: type I, type II and type III. Type II CRISPR systems are the most well understood, and the term "CRISPR" typically refers to these systems and their variants.

In the section above, we made reference to "guide RNAs" conveying the targeting specificity to Cas9 enzymes, and being responsible for guiding the nuclease enzyme to a specific target and introducing a double strand break there. The use of the term gRNA is very much a generalisation, and each CRISPR type has peculiarities relating to the structure, mechanics, and how the gRNA is generated.

For instance, in wild type systems, crRNAs provide the targeting, whereas in genome editing CRISPR systems, a synthetic small gRNA (or sgRNA) serves this role.

 

Example CRISPR vector schematic. A typical CRISPR vector houses a nuclease (e.g. Cas9) a guide RNA cassette (which a guide is cloned into), and a selectable marker. © 2016 Oxford Genetics

Wild-type Type II CRISPR Systems

In wild type II CRISPR systems, the guide RNA is referred to as a CRISPR targeting RNA, or crRNA, and it is transcribed from the DNA sequences known as protospacers, ~20 base pair long sections of foreign DNA separated by a short palindromic repeats in the bacterial genome. To create the crRNA, the entire protospacer array is transcribed as pre-crRNA, and is then cut up into individual crRNAs by a trans-activating crRNA (tracrRNA) that has a sequence complementary to the palindromic repeat. When the tracrRNA hybridizes to the short palindromic repeat, it triggers processing by the bacterial double-stranded RNA-specific ribonuclease, RNase III. The pre-crRNA is then cut into crRNAs, which bind to the tracrRNA, which binds to the Cas9 nuclease, which then becomes activated and specific to the DNA sequence complimentary to the crRNA.

 


An overview of the endogenous Type II bacterial CRISPR/Cas system. Within the bacterial genome, a CRISPR array contains many unique protospacer sequences that have homology to various foreign DNA (e.g. viral genome). Protospacers are separated by a short palindromic repeat sequence. (A) The CRISPR array is transcribed to make the pre-CRISPR RNA (pre-crRNA). (B) The pre-crRNA is processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit the RNAse III and Cas9 enzymes, which together separate the individual crRNAs. (C) The tracrRNA and Cas9 nuclease form a complex with each individual, unique crRNA. (D) Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complimentary to the crRNA. In the Type II CRISPR system a potential target sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly after where the crRNA would bind. (E) After the complex binds, the Cas9 separates the double stranded DNA target and cleaves both strands after the PAM. (F) The crRNA:tracrRNA:Cas9 complex unbinds after the double strand break. © 2014 Addgene

Modified Type II CRISPR Systems for Genome Editing

 


Single guide RNAs are an elegant solution that let a single RNA construct bind to a Cas protein and direct it to a specific target © 2012 Programming Cas9 with single-guide RNAs (sgRNAs) Jinek et al. Science 337, 816 (2012)
The majority of CRISPR systems used in genome editing are modified versions of the naturally-occurring Type II CRISPR system. In these genome editing CRISPR systems, the guide RNA is referred to as a synthetic small gRNA (sgRNA). sgRNAs are transcribed from a genome editing vector (plasmid) which is inserted into a cell. The vector contains the code for a ~20 base pair protospacer, the Cas9 enzyme itself, and a constant scaffold region which provides the functionality of the tracrRNA.

The main difference between genome editing CRISPR systems and wildtype systems is that the sgRNA is a single construct; there is no need to have both the crRNA and a tracrRNA. This is because the protospacer and the scaffold are transcribed as a single unit, resulting in a single sgRNA complex that has the functionality of the tracrRNA (it binds to the Cas9), and the protospacer (it targets a specific region of DNA).

This makes genome editing CRISPR systems a little easier to use, as the number of steps required to build the functional ribonucleoprotein is reduced in the cell. Further, unlike wildtype systems where the bacterial genome is running the code for the CRISPR system off of its own genome, in this case, the code is executed from a vector, which is inserted into the cell by a scientist (see above).

Simply put, the DNA for the Cas9 is transcribed and translated by the cell, and the protein complexes with the guides that are expressed from the protospacers encoded onto the plasmid/vector. 

Generally, genome editing CRISPR applications involve a single Cas9 enzyme from Streptococcus pyogenes known as SpCas9 (with a "NGG" PAM recognition sequence), along with a single synthetic guide RNA (sgRNA). However, different Cas9 enzymes can be used, each with their own PAM sequences, and many CRISPR vectors can in fact have more than one protospacer sequence on it, meaning that multiple genes can be targeted by a single vector that expresses a Cas9 and multiple sgRNAs.

More details of designing of sgRNA targeting a specific region of DNA can be found here

What do you think?

About the author

View full profile Jérôme Lutz from Berlin & Munich, Germany

I like to share the great things I discover daily while researching and working in the field of Synthetic Biology.

When I talk to people about it, they often refer to Science Fiction. However, when I send them links to this wiki and they read through those pages, they start understanding that this is real and it's happening right now.

Share
Follow