Since the introduction of the groundbreaking CRISPR/Cas technology, it has received widespread acclaim and applause. In just a brief two or three years, it has emerged as the most sought-after research tool in the field of life sciences.
So, what exactly is this CRISPR/Cas technology that researchers hold in such high regard?
The CRISPR/Cas system is an adaptive immune system discovered in prokaryotes, comprising Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas) – hence the name CRISPR/Cas system.
Let's delve into the various proteins within the CRISPR/Cas system. Cas is the abbreviation for CRISPR-associated proteins. The CRISPR system encompasses various common Cas proteins, ranging from Cas1 to Cas10, as well as rarer types like Cas8a1, Cas12a, Cas13a, etc.
The CRISPR/Cas system is categorized into two major classes and five types based on the sequence similarity between Cas genes and site structures:
- The first-class systems (including types I, III, and IV) are found in bacteria and archaea, typically forming multi-subunit protein–crRNA (CRISPR RNA) effector complexes.
- The second-class systems (including types II, V, and VI) rely on a single crRNA-guided protein for target interference, performed by a single multidomain protein.
In simpler terms, the classification of class I and class II CRISPR/Cas systems depends on whether the structure of their effector molecules is particularly simple. The effector molecules of class II CRISPR/Cas systems, such as Cas9, Cas12, Cas13, and Cas14, are standalone proteins capable of independently cleaving DNA/RNA.
Among all Cas proteins, Cas1 constitutes 20.11%, Cas2 17.46%, Cas3, Cas5, Cas6, and Cas7 together account for over 10%. The most renowned Cas9 protein makes up only 2.59% of all Cas proteins. This article will focus on discussing Cas9, Cas12, Cas13, and Cas14, which are effector proteins capable of independently cleaving DNA/RNA.
Cas Family Proteins:
Cas1 and Cas2 proteins are core components in the CRISPR system, participating in the integration of exogenous fragments in the initial phase of the immune process. Cas3 possesses DNA cleavage and helicase activities, serving as the core enzyme in type I CRISPR/Cas systems. Cas4 is involved in the process of inserting exogenous DNA sequences into bacterial DNA, primarily found in II-B and I-A/B/C/D type CRISPR/Cas systems. Cas5 protein, along with other proteins, forms the Cascade complex to exert functions. In E. coli (I-E type), the generated crRNA-Cas6 complex recruits 1 Cse protein, 2 Cse2 proteins, 6 Cse7 proteins, and 1 Cas5 protein. Cas6 is responsible for processing pre-crRNA into mature crRNA. Cas7 and Cas8 are both essential components of the Cascade complex.
Cas10 is responsible for cleaving DNA strands and serves as the core enzyme in type III CRISPR/Cas systems. Cas10, together with multiple Csm/Cmr subunits and a crRNA (CRISPR RNA), assembles into an effector complex. This complex can degrade both RNA and DNA (ssDNA). Cas11 mainly participates in crRNA binding and is common in type III CRISPR/Cas systems.
In addition to the mentioned Cas proteins, Cas9, Cas12, Cas13, and Cas14 are core single enzymes in class II systems, widely applied in gene editing technologies. Let's delve into each one:
Cas9 is a DNA endonuclease, and its cutting activity is guided by guide RNA molecules containing sequences complementary to the target DNA, known as PAM sequences. Cas9 precisely cleaves the target DNA three nucleotides upstream of the PAM, forming blunt-ended products. The HNH domain of Cas9 is responsible for cutting the DNA strand complementary to crRNA, while the RuvC domain cuts the non-complementary strand. Ultimately, under the action of Cas9, double-strand DNA breaks (DSB) occur, and the cell utilizes homologous recombination or non-homologous end joining to repair the broken DNA, achieving knockout or knock-in of DNA fragments.
Cas9 has two cutting-active domains: HNH and RuvC. The HNH domain cuts the DNA strand complementary to crRNA, while the RuvC domain cuts the non-complementary strand. By mutating only the RuvC I domain of Cas9, specifically converting one of the two key amino acid residues in RuvC I to alanine (D10A or H840A), a Cas9 nickase (Cas9n) is obtained. This nickase cannot cut the non-complementary DNA strand but can cut the DNA strand complementary to crRNA, significantly reducing off-target effects while maintaining efficient gene modification.
By point mutations that inactivate both active domains, RuvC- and HNH-, where the aspartic acid at position 10 of the RuvC catalytic domain is mutated to alanine (D10A), and the histidine at position 840 of the HNH catalytic domain is mutated to alanine (H840A), a Cas9 protein loses its endonuclease activity, forming dCas9. dCas9 can bind to the target gene under the guidance of sgRNA but lacks the ability to cut DNA. Therefore, binding dCas9 to the transcription start site of a gene can block the initiation of transcription, inhibiting gene expression. Binding dCas9 to the promoter region of a gene can also recruit transcriptional repression/activation factors, suppressing or activating downstream target gene transcription. The difference between dCas9 and Cas9 or Cas9n is that the activation or inhibition caused by dCas9 is reversible and does not result in permanent changes to the genomic DNA.
CRISPR–Cas12a protein, formerly known as Cpf1, is an endonuclease that can be programmed by guide RNA to target complementary DNA sequences. Upon binding to the target DNA, CRISPR–Cas12a protein induces cleavage on each target DNA strand, resulting in double-strand DNA breaks. In addition to inducing cis cleavage of the target DNA, target DNA binding also induces trans cleavage of non-target DNA. Therefore, the CRISPR–Cas12a-RNA guide complex can provide sequence-specific immunity against invasive nucleic acids such as phages and plasmids. Similar to CRISPR/Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. Furthermore, its trans cleavage activity has been applied to highly sensitive nucleic acid detection. The off-target rate of the CRISPR/Cas12a system is much lower than that of Cas9, and because of its differences from Cas9, it can complement with Cas9, expanding the toolbox of CRISPR/Cas systems, making gene editing technologies based on CRISPR/Cas systems more versatile and powerful.
Cas12b is an RNA-guided endonuclease that, in the presence of a PAM on the target DNA, can specifically cleave the target double-stranded DNA. Cas12b is smaller than Cas9 and Cas12a, and it has higher cleavage activity, originating from the acidothermophilic bacterium Alicyclobacillus acidoterrestris. Cas12b's optimal cleavage reaction temperature is 48 ℃. Similar to Cas12a, Cas12b recognizes the PAM sequence 5'-TTN, and it produces sticky ends after cutting the DNA. However, Cas12b is double RNA-guided, depending on both crRNA and tracrRNA (or sgRNA formed after connection). Additionally, Cas12b can be used not only for cutting target dsDNA but also for rapid detection of target nucleic acids, as seen in the HOLMESv2 nucleic acid rapid detection technology.
Cas13 proteins can be further divided into four subtypes: CRISPR-Cas13a, b, c, and d. While research on Cas13c is currently not sufficient, the other three Cas proteins have been widely researched and applied.
Cas13a is an RNA-guided RNA endonuclease. In the presence of a PFS sequence on the target single-stranded RNA, it can specifically cleave the target RNA. Additionally, Cas13a has reverse cutting activity dependent on the target single-stranded RNA, used for developing rapid detection assays for target nucleic acids. For example, researchers at MIT, led by Feng Zhang, have developed a next-generation molecular diagnostic system called "SHERLOCK" using Cas13a protein. For bacteria, Cas13a's reverse cutting activity is meaningful. If bacteria are infected by phages, it can activate programmed cell death or dormancy to limit the spread of infection throughout the population. Similar to Cas13a, Cas13b requires only a single guide RNA to target specific sequences. Moreover, Cas13b can simultaneously target multiple RNA transcripts.
The later-discovered Cas13d has higher efficiency, lower off-target rates, and, importantly, a smaller size compared to other Cas13 family members. Cas13d is more easily packaged into viral vectors, providing better delivery advantages and application prospects.
In addition to the aforementioned Cas proteins with target cleavage capabilities, the recently discovered Cas14 also possesses similar functions.
Cas14a is an RNA-guided endonuclease, which, under the guidance of tracrRNA:crRNA (or sgRNA), specifically binds and cleaves the target ssDNA without the need for a PAM site. Cas14 proteins generally have smaller molecular weights (400-700 aa) compared to other Cas proteins. Similar to Cas12, Cas14a can also bind to target nucleic acids and activate its ssDNA reverse cutting activity. Researchers believe that Cas14's cutting target is single-stranded DNA, not double-stranded DNA, so it might not be an ideal genome editing tool. However, they consider Cas14 useful in the DETECTR diagnostic tool.
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