Restriction Enzymes

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Each restriction enzyme recognises a different and specific recognition site, or DNA sequence. Recognition sites are usually only short - 4-8 nucleotides.

See below as to how CRISPRs differ

Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

These enzymes are found in bacteria and archaea and provide a defence mechanism against invading viruses.  Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction; while host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system.

.<img src="/84e9beda-d57c-5f8b-ac2c-bfedf7b98f54.png"/>       Structure of the homodimeric restriction enzyme EcoRI (cyan and green cartoon diagram) bound to double stranded DNA (brown tubes).

Is a restriction endonuclease any different than a CRISPR?

A restriction enzyme recognizes specific sequence (unique to every enzyme) and makes a cut. For example, EcoRI recognizes the nucleotide sequence GAATTC and cuts it so that it becomes two fragments with overhangs as shown in the following image

Note that no nucleotide is deleted/added. If the sequence is relegated, it is again the same sequence.

For restriction digestion, all you need is a DNA, an enzyme and specific buffer conditions.

However, things are a little bit complicated in CRISPR/Cas9. Here, you can choose any site in the DNA provided some criteria are satisfied like a PAM sequence (required for Cas9 recognition, nGG, n=any nucleotide).

This is how it goes

1) choose a target site

2) design a polynucleotide Guide RNA containing complementary bases of your target site (the part of Guide RNA binding to the target DNA) and the rest of the sequence is required for proper conformational folding for recognition by Cas9 enzyme (blue bubble).

3) Inject the embryos/treat the cells (I work with frog embryos) with (a). guide RNA, (b). Cas9 enzyme (either mRNA or protein).

Once the guide RNA binds to the target sequence, the cas9 enzyme recognizes the site and makes a double strand break in the DNA sequence just before the PAM sequence. This results in INDELS (insertions/deletions). SO the sequence is not the same after break which results in complete loss of the gene function (knockout).

This happens via homologous recombination or non-homologous end joining during cell cycle to repair the damage.

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.

Creating artificial REs.  Feb 2017

Scientists at the University of Illinois say they have developed a new technique of genetic engineering for basic and applied biological research and medicine. Their work ("Programmable DNA-Guided Artificial Restriction Enzymes"), reported in ACS Synthetic Biology, could open new doors in genomic research by improving the precision and adherence of sliced DNA, according to the investigators.

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research; however, they have limited sequence specificity and availability. The Pyrococcus furiosus Argonaute (PfAgo)-based platform for generating artificial restriction enzymes (AREs) is capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length.  CRISPR/Cas9, a bacterial immune system used for "cut-and-paste" gene editing, and TALENs, or transcription activator-like effector nucleases, which are modified restriction enzymes, are two popular examples of such techniques. The new method creates AREs through the use of an Argonaute protein (PfAgo) taken from Pyrococcus furiosus, an archeal species. Led by a DNA guide, PfAgo is able to recognize much longer sequences when finding its cut site, increasing specificity and removing much of the obstacles posed by restriction enzymes. Furthermore, PfAgo can create longer sticky ends than even restriction enzymes, a substantial benefit as compared to other AREs. Historically ARES were very short strands, 4-8 bases, but these new ones are much longer.

Selecting a RE--  see Selecting a restriction enzyme. Summary

The following technique can be used to easily move any piece of DNA from one vector to another as long as it is already bounded by restriction sites that are also present in the same orientation on your target vector. If you are not sure what vector to use, you can check out our Empty Backbone Reference.

Background

Subcloning by restriction digest is a commonly used lab technique. For the purposes of this tutorial we will discuss how to move a cDNA from one plasmid to another. However, the same technique can be used to move promoters, selectable markers, or any other DNA element between plasmids.

Let's assume that you are beginning a new project on your gene of interest (YGOI for short). You might need to express YGOI in cultured mammalian cells. The problem is that the only version of full-length cDNA you can find for YGOI is in a bacterial expression vector. Using subcloning, you can easily move YGOI into a mammalian expression vector.

Restriction Enzymes

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