The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems in bacteria has been a game-changer for gene editing due to their ability to use a guide ribonucleic acid (RNA) and target specific gene sequences.
In a recent review published in Molecules and Cells, researchers discussed the features of various CRISPR-Cas systems, including CRISPR-associated transposons (CASTs) and the eukaryotic equivalent of CRISPR-Cas systems, Fanzor systems.
Study: Genome editing using CRISPR, CAST, and Fanzor systems. Image Credit: Prostock-studio/Shutterstock.com
CRISPR-Cas Systems
The CRISPR-Cas system was first discovered in Escherichia coli, and since then, 90% of genomes from archaea and close to 40% of other bacterial genomes have reported the presence of various CRISPR-Cas systems. It evolved in bacteria and archaea as a defense mechanism against invading plasmids or phages.
Portions of foreign nucleic acids, such as viral deoxyribonucleic acid (DNA), are merged into the CRISPR array as spacers during the pathogen's initial invasion.
These spacers carried inside guide CRISPR ribonucleic acid (crRNA) to help identify these spacer sequences in the pathogens during subsequent invasions and target the Cas proteins to that site to degrade the exogenous genetic material.
This ability to use a guide RNA to perform gene editing at a targeted site was harnessed for genome editing. The CRISPR-Cas system revolutionized gene editing since it could be modified to target any sequence by manipulating the crRNA.
Based on the number of Cas proteins, there are two classes of CRISPR-Cas systems. Class 1 consists of multiple Cas proteins and CRISPR, while Class 2 has only one Cas protein. Each of the two classes consists of three types of CRISPR-Cas systems.
Analogous systems known as obligate mobile element-guided activity or OMEGA systems, such as the Fanzor system in eukaryotes, have also been discovered. They exhibit similar guided nuclease activity.
Types of CRISPR-Cas Systems
The most used CRISPR-Cas system in gene editing is the CRISPR-Cas9 system, which is a type II system belonging to Class 2. The first Cas9, SpCas9, was obtained from Streptococcus pyogenes. It was observed that SpCas9 recognized an NGG (where N stands for any nucleotide and G stands for guanine) motif as the protospacer-adjacent motif or PAM.
It also required two RNAs — a crRNA and a trans-activating crRNA called tracrRNA, which formed a dual guide with crRNA. Orthologs of SpCas9 that recognize different PAMs have since been discovered in Campylobacter jejuni, Neisseria meningitidis, and Neisseria cinerea.
Unlike CRISPR-Cas9, the CRISPR-Cas12 system requires only one guide RNA and recognizes a TTTN (where T stands for thymine) sequence as the PAM.
The off-target effects of CRISPR-Cas12 are lower, and variants of Cas12, such as the Cas14a protein from archaea, do not require a PAM sequence to cleave. The CRISPR-Cas12 system is also a Class 2 system of type V.
Another Class 2 CRISPR-Cas system is the type VI CRISPR-Cas13 system, which differs from the previous two in its ability to target RNA instead of DNA. Multiple subtypes of Cas13 can collaterally cleave RNA and have been used for highly sensitive viral detection.
OMEGA Systems
The review also presented some analogous gene editing systems, such as CAST, which integrates the Tn7-like transposon comprising the transposon proteins TnsA, TnsB, TnsC, TnsD, and TnsE.
In CAST, the Tn7-like transposons are integrated into the DNA using the CRISPR-Cas12k system, which does not have nuclease activity. This allows large fragments of DNA to be inserted without inducing double-stranded DNA breaks.
Although the CAST from Scytonema hofmanni can insert DNA fragments as large as 10 kilobase pairs, it also comes with a high risk of off-target effects. However, the CAST from Vibrio cholerae can insert similarly large DNA fragments with a lower risk of off-target effects.
The researchers also discussed the similarities between ancestral proteins such as transposon-associated ribonucleoprotein TnpB and the transposon-encoded IscB protein, which could evolutionarily be linked to Cas12 and Cas9, respectively.
The Fanzor system in eukaryotes is believed to have evolved from TnpB protein and exhibits CRISPR-Cas-like activity. However, it uses non-coding RNA to form the nucleoprotein complex and targets and cleaves specific motifs within eukaryotic cells.
Applications of CRISPR-Cas Systems
After the initial in vitro experiments using CRISPR-Cas systems, the technology has been applied to various animal models such as zebrafish, fruit flies, rodents, and Caenorhabditis elegans to examine its in vivo efficacy and uses.
These in vivo experiments have furthered our understanding of human genetics and diseases and helped improve therapeutic options for numerous genetic disorders.
Conclusions
The CRISPR-Cas system has significantly improved over traditional gene editing techniques, such as transcription activator-like effector nuclease and zinc-finger nucleases, by providing an easily modifiable method of targeting specific gene sequencing for editing.
Furthermore, the wide variety of CRISPR-Cas systems and their unique features also broaden its scope of application. The discovery of OMEGA systems in other groups is also believed to increase the versatility of CRISPR-Cas systems in future applications.
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