CRISPR/Cas9(Clustered Regulated Interspaced
Short Palindromic Repeats) is a major
breakthrough in gene editing. Many scientists believe that CRISPR Cas9 is the
most effective and efficient tool to perform such experiments.
use it to edit bacterial genomes in order to deal with epidemic illnesses
caused by bacteria and to increase crop yield. There are already many species
of bacteria that have been successfully edited with CRISPR/Cas9.It can, for
instance, regulate the bacterial virulence and be an indicator of antibiotic
resistance to some pathogenic bacteria. CRISPR/Cas itself has modernized the
classification and evolution mapping of bacteria.
However, there are
several challenges it faces in some species. Other speciesstill fail to be
edited due to incompatibility with CRISPR Cas itself, in parts because of a
targeted bacterium’s instability and because CRISPR/Cas could be toxic to some
It is to be expected
that CRISPR Cas will still be able to be improved in such a way that could
innovate the world of molecular biology, however not as soon as ten years into
the future, as originally predicted.
The overall aim of this literature review was to produce a comprehensive
literature paper which focused on CRISPR/Cas and finding successfully genome
edited bacteria using Cas9 as well as addressing the problems and challenges
CRISPR/Cas9 has faced.
years ago, scientists began to delve deeper into the structure of bacterial DNA
and a scientist, Francisco Mojica
discovered CRISPR (Clustered Regulated Interspaced Short Palindromic Repeats).
In 2005, Alexander Bolotin found Cas9
PAM (Protospacer-adjacent motifs). Between 2005 and 2013, many scientists made contributions
that made the CRISPR/Cas9 system possible. By 2011, tracrRNA for the
Cas9system was discovered by Emmanuelle CharpentierandCas9-mediated cleavage was characterized
biochemically by Virginijus Siksnys.By 2013,CRISPR-Cas9 was successfully applied in genome editing in
eukaryotic cells for the first time by
Feng Zhang. CRISPR has
exploded in the last four years, seeing exponential growth, and eventually been
reported by Emmanuelle Charpentier
and Jennifer Doudna by 2013. The
history of CRISPR/Cas is long and full of work done by many commendable
scientists and the future itself is bright.
CRISPR Associated Protein or CAS Protein
proteins (Cas proteins) have two roles. The first one is their use of stored
sequence information to identify viruses, or foreign genomes and destroy them.
The second is its involvement in obtaining and storing segments of a virus
There are different
types of CRISPR systems (types I-III) and the Cas proteins are
typically adjacent to the CRISPR system and serve as a basis for the
classification of three different types of CRISPR systems. Types I and III
CRISPR systems contain multiple Cas proteins, whereas the type II system
mostly uses Cas9 proteins. Since the initial studies, the CRISPR-Cas9
system has been used by thousands of laboratories for genome editing
applications in a variety of experimental systems.
Cas9 is paired with the CRISPR system type II which is mostly found in
bacteria of the genus Streptococcus. One of the most widely known Cas proteins
that are being used is the Streptococcus phyogenes Cas9 (spCas9). The
Cas9 protein is one of the most important components for engineering genomes.
The Cas9 protein binds to the crRNA/tracrRNA hybrid which acts as a guide for
the protein. The protospacer encoded portion of crRNA directs the Cas9 protein
to cleave complementary target DNA sequences, if they are adjacent to the short
sequences known as protospacer adjacent motifs (PAM).
The CRISPR defence system requires the
transcription of the repeat-spacer array from a leader sequence that acts as a
promoter, and is used in conjunction with an RNA-processing system containing
eight genes, called Cas genes (CRISPR-associated). In E. Coli, these genes are called K12, and are usually located
adjacent to each CRISPR locus. Cas genes
code for a variety of RNA-binding proteins, polymerases and nucleases (both DNA
and RNA). There are three major families of CRISPR/Cas genes, depending on the
specific Cas proteins in the genome. There is a multimeric complex called Cascade (CRISPR-associated complex for
anti-viral defence) composed of five Cas
proteins and is responsible not only for the interference stage, but also for
the adaptation stage, which processes the foreign invader for incorporation
into the CRISPR locus.
CRISPR region is transcribed into a long RNA
(pre-crRNA) which is processed into short CRISPR RNAs composed of about 57
nucleotides containing a spacer flanked by two conserved partial repeats, the
PAMs (protospacer-adjacent motifs). These spacer/PAM RNAs that are complementary
to phage DNA protospacer sequences, are subsequently used as guides for the Cas interference machinery. Pairing is
initiated by a high-affinity seed sequence at either end of the crRNA spacer
The complex base matches with the virus DNA or
RNA to prevent expression of the phage genes and by last leads to degradation.
Mutations in either the spacer DNA core seed sequence or the PAM sequence
annuls CRISPR/Cas immunity by altering binding. These mechanisms offer powerful
approaches for turning off genes at will and altering gene expression. Though
it is not necessarily a one-way where a regulatory RNA is produced and turns
off expression of a message. This method can also be balanced by the production
of a counter protein that can link to and interfere with the sRNA. Dynamic
systems can exist that can change over time,per cell demands.
1. Stage I:
to do with the entry of foreign DNA into a cell through transformation,
conjugation, or transduction which can lead to acquisition of new DNA spacer(s)
by the adaptation Cas complex (unknown protein assembly). If no spacer is
acquired, the phagelytic cycle or plasmid replication can proceed.
2. Stage II:
The interfering Cas
complexes are bound to a crRNA produced from the transcription of the CRISPR
locus and subsequent processing. A cell carrying a crRNA targeting a region (by
peect pairing) of foreign nucleic acid can interfere with the invasive genetic
material and destroy it via an interference Cas complex (unknown protein
assembly except for Cascade in Escherichia coli). If there is no perfect
pairing between the spacer and the protospacer (as in the case of a phage
mutant), the CRISPR/Cas system is counteracted and replication of the invasive
genetic mate al can occur.