•  Home
•  Products
•  Life Science
•  Enzymes
•  Class 2 CRISPR/Cas Enzymes

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) -CRISPR-associated (Cas) system is an adaptive immune system in prokaryotes that protects against phage infection by storing memories in the form of viral DNA in the bacterial host chromosome. The system contains viral DNA surrounded by repetitive nucleotide sequences called direct repeats. These direct repeats are surrounded at the proximal end by sequences encoding the Cas proteins. Through a process called adaptation, bacterium capture snippets of foreign genetic elements and incorporate them into their genomic CRISPR-array. Transcription of the CRISPR array produces CRISPR-RNA (crRNA) that binds to the CRISPR endonuclease Cas protein and provides specificity through base-pairing with the target nucleic acid.

CRISPR-Cas system is artificially manipulated in guiding reprogrammed endonucleases to the target gene. CRISPR genome editing technology modifies internal DNA/RNA in a sequence-specific way and is reprogrammable. CRISPR technology has been successfully applied in the fields of agriculture, therapeutics, the food industry, and bioenergy.

CRISPR-Cas systems are divided into 2 classes, 6 types, 33 subtypes, and several variants. The class 1 system consists of the type I, type III, and type IV systems. This class utilizes effector complexes consisting of multiple Cas proteins and crRNAs in the interference step. The class 2 system consists of the type II, type V and type VI systems. Unlike Class 1 CRISPR–Cas systems, the effector complex of Class 2 CRISPR–Cas systems is a single, large, multi-domain Cas protein that binds to crRNA.

Class 2 CRISPR/Cas Enzymes

CRISPR-Cas9 System

CRISPR-Cas9 system has been the first and most widely adopted for genetic engineering, which contains Cas9 protein, crRNA, and trans-activated CRISPR-RNA (tracrRNA). Properties of the endonuclease Cas9 protein ensure accurate and efficient editing. By specifically recognizing the crRNA and its interaction with a tracrRNA, Cas9 assembles with the specific guide RNA. In addition, the dual crRNA-tracrRNA could be fused into a chimeric single guide RNA (sgRNA) to create a two-component system, Cas9 and its sgRNA. Finally, stable binding of the target DNA adjacent to the protospacer adjacent motif (PAM) with the correct nucleotide sequence acts as a switch to trigger Cas9 to introduce a dsDNA break. Cas9 has a switchable nuclease activity and can be easily redirected by altering the sgRNA targeting region.

SpCas9

SpCas9 is a DNA endonuclease derived from S. pyogenes and specifically recognizes and cleaves dsDNA target in a PAM (NGG)-dependent manner. The cleavage site of Cas9 in the target sequence is 3 bps away from the PAM site. In addition, SpCas9 can also specifically cleave ssDNA or ssRNA in the presence of DNA PAMmer sequences.

Sp-dCas9

The HNH and RuvC domains of SpCas9 are responsible for cleaving the target and non-target strands of dsDNA, respectively. In Sp-dCas9, both H840 and D10 are mutated to A, so that its HNH and RuvC domain are inactivated. Sp-dCas9 has only the target DNA binding activity, but not the cleaving activity.

Sp-nCas9

In Sp-nCas9 (also called SpCas9 H840A Nickase), the H840 residue in Cas9 is mutated to A, thereby inactivating its HNH domain. Therefore, only the RuvC domain in SP-nCas9 has the cleavage activity, cleaving the NT strand in the dsDNA target to generate a nicked dsDNA product.

CRISPR-Cas12 System

Among Class II Type V CRISPR-Cas systems, Cas12a (Cpf1) was the first to be identified, followed by Cas12b (C2c1) and other type V (Cas12) systems. The Cas12 systems exhibit great diversity and share several common features that differ from Cas9 systems. Cas12 nucleases possess a RuvC nuclease domain but no HNH domain, and they recognize the T-rich PAM 5 'upstream of the target region on the untargeted strand, unlike the Cas9 system, which relies on the G-rich PAM at the 3' side of the target sequence. Cas9 generates a blunt end proximal to the PAM sequence, but Cas12 generates staggered double‐stranded breaks (DSBs) distal to the PAM. The staggered DSBs may support a unique targeting strategy for gene knock-in through the non-homologous end joining (NHEJ) mechanism. Despite these shared properties, Cas12 systems present enormous structural and functional diversities.

Cas12a

Cas12a (previously known as Cpf1) is a DNA endonuclease guided by crRNA alone. Cas12a can specifically cleave dsDNA target in a PAM-dependent manner, generating DSBs, and cleave ssDNA target in a PAM-independent manner. In addition, both dsDNA and ssDNA targets can trigger the trans-cleavage activity of Cas12a. When Cas12a binds crRNA and target DNA to form a ternary complex, the trans-cleavage activity of Cas12a is released to non-specifically cleave ssDNA sequences in the reaction system. Therefore, Cas12a can be used for specific cleavage of dsDNA in vitro and rapid detection of target nucleic acids.

Cas12b

Cas12b (previously known as C2c1) is a DNA endonuclease mediated by tracrRNA and crRNA (or sgRNA), which can specifically cleave the dsDNA target in a PAM (TTN)-dependent manner, generating DSBs, and cleave ssDNA target in a PAM-independent manner. Both dsDNA and ssDNA targets can trigger the trans-cleavage activity of Cas12b.

AacCas12b is derived from Alicyclobacillus acidoterrestris, and its optimal reaction temperature is 48°C. AapCas12b is derived from Alicyclobacillus acidophilus, and its optimal reaction temperature is 60°C. Compared to AacCas12b, AapCas12b is more suitable for “One-Step” CRISPR-based detection systems (CRISPR-Dx) system with the integration of LAMP isothermal amplification.

Cas12f

Cas12f1 (previously known as Cas14a1) is an endonuclease that specifically binds and cleaves ssDNA target under the guidance of tracrRNA and crRNA (or sgRNA alone), and does not require a PAM site. In addition, Cas12f1 can also specifically cleave dsDNA target in a PAM-dependent manner, generating DSBs with sticky ends. Similar to other Cas12 proteins, Cas12f1 has the trans-cleavage activity against ssDNA, and both dsDNA and ssDNA targets can trigger its trans-cleavage activity. When Cas12f1 binds sgRNA and target DNA to form a ternary complex, it releases trans-cleavage activity to non-specifically cleave ssDNA sequences in the reaction system. Cas12f1 protein is generally smaller (400-700 AA) than other Cas proteins and can be used to develop CRISPR-Dx systems for rapid detection of nucleic acids.

Un1Cas12f1

CRISPR-Cas13 System

Type VI CRISPR-Cas family members include Cas13a, Cas13b, Cas13c and Cas13d. Unlike Cas9 and Cas12, the Cas13 proteins have the unique property of cleaving ssRNA but not DNA. The subtypes of the Cas13 lack a DNA catalytic domain, which is replaced by two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains, each containing an RNA cleavage site.

The members of the CRISPR-Cas13 system are dual‐component systems in which a crRNA forms a complex with the Cas13 protein without any involvement of tracrRNA. The flanking sequence of the protospacers, called protospacer flanking site (PFS) and comparable to the PAM of Cas9 and Cas12, is essential for the RNA targeting process. A distinctive feature of the Cas13 system is the collateral cleaving activity towards non‐targeted, unspecific RNAs in the reaction environment. Upon binding to the target RNA, the catalytic pocket formed by the two HEPN domains is activated and can cleave exposed RNA indiscriminately in solution, including endogenous RNA of housekeeping genes. Thus, CRISPR‐Cas13 systems are potential tools for RNA editing.

Cas13a

Cas13a (previously known as C2c2) is a crRNA-mediated RNA endonuclease that can specifically recognize and cleave the RNA target with the PFS sequence. When Cas13a binds crRNA and RNA target to form a ternary complex, it releases the trans-cleavage activity of Cas13a against non-target ssRNA sequences. This activity of Cas13a has been applied in the development of rapid nucleic acids detection systems.

LwaCas13a