1: Introduction

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What is CRISPR/Cas?

The CRISPR/Cas system is the prokaryotic immune system. When a virus or other foreign attacker attempts to infect a prokaryotic cell and inject its own DNA into a prokaryote’s genome, the prokaryote’s CRISPR/Cas system is responsible from removing the foreign DNA. How does it do this? The CRISPR/Cas system has two parts, CRISPR and Cas. The CRISPR part (a CRISPR array), is responsible for ”remembering” the foreign DNA, while the Cas part (Cas proteins), is responsible for cutting out the recogined foreign DNA. A CRIPSR array is made up of segments of short spacer DNA, which are the results of previous exposure to foreign DNA. These spacer DNA are transcribed to RNAs, which can be used to match the foreign DNA that the spacer DNA was built from. These RNA are then picked up by Cas proteins. When a Cas protein picks up a particular RNA, it becomes sensitive to the matching DNA sequences. The next time the same foreign DNA is inserted into the prokaryote, the Cas proteins sensitive to it will match the foreign DNA and cut it out of the genome, causing it to become inactive.

Why is CRISPR/Cas important to us?

Because nature is giving us an effective way of editing a genome! In order to accurately edit a genome, it is important to be able to cut a sequence at precisely the targeted location. Once a cut is made, repair mechanism can go in and make a modification at the target site. The CRISPR/Cas system is a naturally occurring time tested method of doing making alterations to DNA sequences.

Currently, the ability of researchers to perturb and interrogate the genome is lagging behind the current level of techniques for reading. CRISPR provides an effective way to write to the genome that we are capable of reading, allowing us to determine what variations in the genetic code give rise to diseases of interest.

Cas-9

The CRISPR/Cas-9 system is a system that has been of particular interest. Cas-9 is a endonuclease that can trigger gene repair by making cuts at specific target sites, guided by a 20-nucleotide sgRNA. When a target site that is complementary to the guide sgRNA is found and is followed by a NGG PAM region, the Cas-9 protein will cut the DNA at that target site. By programming Cas-9 with specific sgRNA, it can be programmed to create double stranded breaks at specific targets, while the PAM region plays a role in prevent targeting of its own genome. Cas-9 has been shown to be much more ecient at targeting than more established methods. Unfortunately, one drawback of Cas-9 is that it might make cuts at off-target sites that aren’t fully complementary to the RNA guide, which makes it a challenge for accurate genome editing.