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Crispr-cas as a New Tool for Genome Editing

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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.

Nowadays, scientists 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 species still 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 bacteria.

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.

CRISPR/Cas9 System History

Twenty-three 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

CRISPR associated 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 sequence.

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 pyogenes 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).

CRISPR/CAS Mechanism

The CRISPR defense 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 defense) 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 sequence.

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.

The mechanism of CRISPR/Cas (Richter, Chang and Fineran, 2012):

  • Stage I: Adaptation.
  • This is 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.

  • Stage II: Interference.

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 perfect pairing) of foreign nucleic acid can interfere with the invasive genetic material and destroy it via an interference Cas complex (unknown protein join 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.

Importance of bacteria and their function in the CRISPR/Cas system

Escherichia coli played a big role in the discovery of the CRISPR/Cas system, since it was the first bacterium in which researchers discovered repetitive DNA sequences which later proved to be part of the bacterium’s CRISPR/Cas system. First considered junk DNA, researchers found that this CRISPR-DNA was part of a prokaryotic equivalent of an immune system that protects bacteria from viral infections.

The principle behind the CRISPR-Cas system is akin to what is known as RNA interference in eukaryotic cells.

In bacteria of the strain Streptococcus pyogenes, this principle is simplified to the point of only requiring two RNA molecules and the enzyme Cas9 for protection. Naturally, the Cas9 protein binds to the crRNA/tracrRNA hybrid which acts as a guide for the protein. This can be replaced by the synthetic sgRNA (single-guided RNA) which does not restrict Cas9’s function and allows for targeted genome editing. The Cas9 protein is mostly found in the Streptococcus pyogenes strain. A smaller version of Cas9 can be found in Streptococcus aureus. The smaller size makes it easier to insert the protein into mature cells and thus overcomes one of the limits of the regular Cas9 enzyme.

Application of CRISPR Cas9

  1. Cas9 as a regulator of bacterial virulence and an indicator of antibiotic resistance.
  2. A large variety of important pathogens of mammals have type II CRISPR-Cas systems, including themajority of pathogens such as L. monocytogenes, S. pyogenes, Streptococcus agalactiae, Neisseria meningitides, C. jejuni, Haemophilus influenza, and Helicobacter pylori. Indeed, the absence or deletion of Cas genes can lead to the increase of antibiotic resistance and uptake of phages. [1]

    However, in another case where there was a lack of a CRISPR-Cas system, asupplemented Cas9 protein led to a significant increase of virulence.

    As is the case for Enterococcus faecalis. The Cas9 gene itself has a role in engineering bacteria, which is to be against conjugative antibiotic resistance plasmid transfer in Enterococcus faecalis (a Gram-positive bacterium).It wasfound that CRISPR-Cas and restriction-modification systems,which function as adaptive and innate immune systems in bacteria, significantly impactthe spread of antibiotic resistance genes in E. faecalispopulations. The loss ofthese systems in high-risk E. faecalissuggests that they are immunocompromised, atrade-off that allows them to readily acquire new genes and adapt to new antibiotics.Mobile genetic elements (MGEs)such as conjugative and mobilizable plasmids and transposons are common in clinical isolates of E. faecalis. They encode resistance to vancomycin, aminoglycosides, tetracycline, chloramphenicol, ampicillin, linezolid, and other antibiotics.

    To provide immunity to MGEs, the CRISPR is transcribed into a pre-CRISPR RNA (pre-crRNA) and processed to mature crRNAs using RNase III, Cas9, and atrans-activating crRNA (tracrRNA) that has sequence complementarity to CRISPR repeats. This is the expression phase. If an MGE possessing the protospacer and PAM (Protospacer adjacent motif) enters the cell, the Cas9 nuclease is directed to the MGE genome by a crRNA/tracrRNAcomplex with sequence complementarity to the protospacer.[2] The HNH endonucleasedomain of Cas9 cleaves the complementary protospacer strand, and the RuvC endonuclease domain of Cas9 cleaves the non-complementary protospacer strand, generatinga double-stranded DNA (dsDNA) break in the invading MGE.This is the interferencephase. In summary, type II CRISPR-Cas systems provide adaptive immunity againstMGEs.[3]

  3. Classification and evolution mapping: via genetic analysis or in vitro experiment.[4]
  4. The evolution of microorganisms is always followed by the numerous changes of their genetic codes and organelles such as the variants of Cas9 types. Since the CRISPR/Cas9 system has been discovered, there is one more way to determine the classification of bacteria. Even if it could complicate and make a big change in the phylogeny tree of bacteria, it could help microbiologists map and rearrange bacteria phyla more precisely.

  5. Editing other bacterial genome from different phyla.
  6. So far, this was successfully used in several Gram-positive (Lactobacillus reuteri) and negative (Francisellanovicida) bacteria, and Cyanobacteria (Synechococcus) with success.

Problems and Challenges

  1. The Cas9 enzyme can be toxic to some bacteria and result in cell death (depending on protein (enzyme) concentration).
  2. Based on the experiment on Cyanobacterium Synechococcuselongatus UTEX 2973, Cas9 proteins can be toxic to its host at some level. The fact is that only five colonies of Synechococcus were yielded from conjugation in a medium containing moderate levels of the Cas9 protein while there were 250 colonies in a medium lacking Cas9. There is no known reason for it, but one possibility is that S. pyogenes Cas9 has off-target effects in cyanobacterialcells. The enzyme may be cleaving genomic DNAin regions other than those targeted by the syntheticsg RNA, and that the cell is unable to repair these breaks thus resulting in lethality. The solution is that the Cas9 expression must occur in a transient manner to achieve successful editing. The fact that editing success depends on transient Cas9 expressionin one Cyanobacterial strain suggests that Cas9 toxicitymay be the reason the application of CRISPR/Cas9genome editing in cyanobacteria has lagged behind that of other organisms.[5]

  3. Off-target effects of CRISPR Cas9 nucleases can cause severe damage. Referring to 1, it is possible to cause bacteria death.
  4. Instability of the bacterial genome can disturb the work of CRISPR/Cas9 to edit a bacteria genome and possibly lead to off-target effects.
  5. Bacterial genome stability is constantly threatened by external agents, such as mobile elements or phages, as well as by the operation of their own DNA replication and repair systems at related or repeated sequences. A growing bacterial population develops a balance between genome maintenance and instability that depends on the type of bacterium, the cell cycle, and the environment. Furthermore, bacteria utilize genome instability to increase their gene diversity and control gene expression and the response to various stresses. The CRISPR/Cas9 as an antibody protects the bacterium from exogenous materials and degrades them. Besides, it can be used to edit bacterial genomes. The instability of bacterial genomes can disturb the work of Cas9 as a part of antibody and editing tool system. [6] In the editing tool case, it would disrupt the function of Cas9 and change its target. This results in undesirable side effects.

  6. Not all bacteria have been yet modified with involving Cas9 protein.


Louwen R, Staals RHJ, Endtz HP, van Baarlen P, van der Oost J. 2014. The role of CRISPR-Cas systems in virulence of pathogenic bacteria.

Cencic, Regina et al. “Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage”. 9.10 (2014):

Price VJ, Huo W, Sharifi A, Palmer KL. 2016. CRISPR-Cas and Restriction-ModificationAct Additively against ConjugativeAntibiotic Resistance Plasmid Transfer inEnterococcus faecalis.

Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJM, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. 2015. An updated evolutionary classification of CRISPR–Cas systems.

Wendt KE, Ungerer J, Cobb RE, Zhao H, Pakrasi HB. 2016. CRISPR/Cas9 mediated targetedmutagenesis of the fast growingcyanobacterium Synechococcus elongatus UTEX2973.

Darmon E, Leach DR. 2014. Bacterial Genome Instability.

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