North South University: CRISPR-Cas9
North South University: CRISPR-Cas9
North South University: CRISPR-Cas9
Assignment-5
CRISPR-Cas9
Group members:
The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign
genetic elements such as those present within plasmids and phages that provides a form of
acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins
recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA.
CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of
sequenced archaea.
Recently CRISPR-Cas9 system has become a potential genome editing tool that enables
geneticists and medical researchers to edit parts of the genome by removing, adding or altering
sections of the DNA sequence.
The CRISPR-Cas9 system consists of two key molecules that introduce a change into the DNA.
These are, 1) Cas9, An enzyme which acts as a pair of ‘molecular scissors’ that can cut the two
strands of DNA at a specific location in the genome so that bits of DNA can then be added or
removed; 2) A piece of RNA called guide RNA (gRNA). This consists of a small piece of pre-
designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The
scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the
genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome. The guide
RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA
bases that are complementary to those of the target DNA sequence in the genome. This means
that, at least in theory, the guide RNA will only bind to the target sequence and no other regions
of the genome. The Cas9 follows the guide RNA to the same location in the DNA sequence and
makes a cut across both strands of the DNA. At this stage the cell recognizes that the DNA is
damaged and tries to repair it.
Figure 1: Mechanism of CRISPR-Cas9 system.
Because genome editing leads to permanent modifications within the genome, the targeting
specificity of Cas9 nucleases is of particular concern, especially for clinical applications and
gene therapy. A combination of in vitro and in vivo assays has been typically used to
characterize the specificity of ZFNs and TALENs (Gabriel et al., 2011), but systematic analysis
has remained challenging due to difficulties in synthesizing large libraries of proteins with
varying sequence specificity. However, Cas9 target recognition is dictated by the Watson-Crick
base-pairing interactions of an RNA guide with its DNA target, enabling experimentally
tractable and systematic evaluation of the effect of guide RNA-target DNA mismatches on Cas9
activity.
To date, the Streptococcus pyogenes Cas9 (SpCas9) has been used broadly to achieve efficient
genome editing in a variety of species and cell types, including human cell lines, bacteria,
zebrafish, yeast, mouse, fruit fly, roundworm, rat, common crops, pig, and monkey. SpCas9 is
also dramatically expanding the catalog of genetically tractable model organisms, for example,
by enabling the introduction of multiplex mutations in cynomolgus monkeys.
Cas9 can be used to facilitate a wide variety of targeted genome engineering applications. The
wild-type Cas9 nuclease has enabled efficient and targeted genome modification in many species
that have been intractable using traditional genetic manipulation techniques. The ease of
retargeting Cas9 by simply designing a short RNA sequence also enables large-scale unbiased
genome editing experiments to probe gene function or elucidate causal genetic variants.
The latest generation of Cas9-based genome engineering tools is also based on components from
the microbial antiphage defense system. It is highly likely that the future solutions for efficient
and precise gene modification will be found in as of yet unexplored corners of the rich biological
diversity of nature.
References