CRISP – Ypatent

Companies: Beam Therapeuticsm Caribou Biociences, CRISPR Therapeutics, Editas Medicine, Intellia Therapeutics, Poseida, Prime Medicine, Sangamo Therapeutics, Wave LifeSciences, Verve Therapeutics

Introduction:

CRISPR Cas systems are a main protective mechanism of bacteria against phages that are prevalent in nature. Although the numbers vary across ecosystems, it is estimated that the ratio of bacterial cells to pahge particles is about 1:10 in may natural environments. Thus, an effective protection against phage infection is crucial for bacterial survival. CRISPR Cas systems are considered as the adaptive immune system of bacteria, since they prevent recurring phage infection by specific degradation of the respective phage genome. Upon first infection with a virus, its genome can be degraded and pieces of the genome can then be integrated into the bacterial chromosome in the form of spacers betwen palindromic repeats form the CRISPR arrays. The mature CRISPR RNA (crRNAs; processing product sonisting of one spacer and one repeat sequence) are mandatory to bind to a specific recognition sequence, the ptrotospacer in the phage genome, leading to an activaiton of nucleolytic activity of the Cas-crRNA complex. (Kretz, “Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus” Frontiers in Microbiology, 2024).

Bacteria have evolved RNA-mediated adaptive defense systems called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protect organisms from invading viruses and plasmids. CRISP/Cas systems are composed of cas genes organized in operons(s) and CRISPR array(s) consisting of genome-targeting sequences (called spacers) interspersed with identical repeats. Bacteria haboring CRISPR loci respond to viral challenge by integrating short fragments of forein sequence (protospaceers) into the host chromosome at the proximal end of the CRISP array. In the expression and interference phases, transcription of the repeat spacer element into precursor CRISPR RNA (pre-crRNA) molecules followed by enzymatic cleavage yeild the short crRNAs that can pair with complementary protospacer sequences of the invading viral targets. Target recognition by crRNAs directs the silencing of the foreign sequences by means of Cas proteins that function in complex with the crRNAs. One important type of CRISPR/Cas system is the type II system where a trans-activating crRNA (tracrRNA) complementary to the repeat sequences in pre-crRNA triggers processing by the double-stranded (ds) RNA specific reonuclease RNase III in the presence of the Cas9 protein. Cas9 proteins constitute a family of enzymes that require a base-paired structure formed between the activating tracerRNA and the targeting crRNA to cleave target dsDNA. Site-specific cleavage occurs at locations determined by both base-paing complentarity between the crRNA and the target protospaces DNA and a short motif. Cas9 is a DNA endonuclease guided by two RNAs. (Jinek “A programmable dual-FNA-guided NA endonuclease in Adaptive Bacterial Immunition” Science, 337, 2012).

About 60% of bacteria and 90% of archaea possess CRISPR (clustered regularly interspaced short palendromic repeats)/CRISPR associated (Cas) system to conver resistance to foregin DNA elements. Doudna (US 10,266,850).

In the adaptation phase, immunological memory is cre-ated by inserting the invading nucleic acid fragment as a new spacer into the CRISPR array. In the expression phase, the CRISPR array, containing variable spacer sequences and interspaced by conserved repeat sequences, is transcribed and processed to yield short CRISPR RNAs (crRNAs). These crRNAs specific to the foreign nucleic acid sequence form an effector complex with CRISPR- associated (Cas) proteins that searches the cell for foreign nucleic acids. Many CRISPR-Cas nucleases require a protospacer adjacent motif (PAM), a short sequence adjacent to the spacer target sequence within the target nucleic acid. Upon Cas–crRNA complex target recognition through base pairing, the complex is activated, and the foreign nucleic acid is cleaved. (Balderston, CRISP Journal, 4(3), 2021).

Critical “spacer” sequences inserted between the repeat elements of nautral CRISPR-cas loci in bacteria and archaea are derived form the genomes of previous invaders, usually viruses and plasmids. The CRISPR spacers thus constitute a genomically recorded memory of past cellular introusions. CRISPR-RNAs (crRNAs) transcribed form these repeat-spacer sequences guide Cas proteins to their targets, and the Cas proteins then initial target desctruction,. There are numerous, mechanistically diverse CRISPR0Cas systems but most of them are RNA guided DNA cleaving pathways and those that use a particular Cas protein (Cas9) have revolutionized biomedical research. (Barrangou, human Gene Therapy, 26(7), 2015).

CRISPR-associated genes (cas) form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences form virulent phages into the CRISPR locus. Subsequently CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. The sequence on the viral genome that corresponds to a spacer is termed “proto-spacer”. CRISPR loci consist of noncontiguous direct repeat sequences that are separated by unique sequences called spacers. These spacer sequences typically derive for foreign genetic elements such as viruses and plasmids. CRISPR repeats and spacers usually vary between 21-48 and 21-72 bp, respectively. The number of spacers within a particular locus can vary widely and reach several hudred. Although repeat sequences can vary across loci, GTTTg/c and GAAAC sequences are common at the 5′ and 5′ end of the repeat, respectively. (Barrangou, “CRISPR: New horizons in phage resistance and strain identification”. Annu. Rev. Food Sci, Technol 2012, 3: 143-62).

Classification of CRISPR Systems:

CRISPR-Cas systems have been divded largely into two classes: class 1 systems, including types I, III and Iv that use multi-protein complexes to destroy foreign nucleic acids nad class 2 systems, including types II, V and VI, that use single proteins. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Enzymes within the CRISPR systems are diverse in their mechanisms. Although all known CRISPR-Cas systems share a conserved mechanism for the adaptation phase, they differ in their interference mechanisms. More specifically, CRISPR systems vary by their effector modules used in the interference phase, which can be either protein subunits or protein complexes bound to a crRNA that orchestrates the cleavage of the target nucleic acid molecule. Recent classifications divided CRISPR enzymes into two classes. Class 1 comprises types I, III, and IV, which all have multi-Cas protein effector modules that associate with the crRNA molecule and bind to the target nucleic acid for subsequent cleavage. Class 2 includes types II, V, and VI, where single multisubunit proteins complex with the corresponding crRNA to mediate the interaction with their specific nucleic acid target. (Balderston, CRISP Journal, 4(3), 2021).

Class 1 CRISPR-Cas systems are most common in bacteria and archaea. (Shmakov Nat Rv Microbiol. 2017, 15(3) 169-182).

Although Class 1 CRISPR systems are the most abundant in bacteria and archaea, gene editing applications are limited by the fact that they have multiple-subunit effectors. Of the class 1 systems, type I is the most widely used for gene editing, especially in created long deletions. During type I mediated gene editing, the CRISPR associated complex for antiviral defense (Cascade), composed of multiple subunit effectors and a crRNA, binds target DNA and forms an R loop structure. Then, Cas3 specifically is recruited and cleaves the target DNA. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Class 2 CRISPR-Case systems: The remaining 10% of CRISPR-cas loci belong to class 2 CRISPR-Cas systems (which use a type II, V or VI effector protein); these sytems are found almost exclusively in bacteria. Class 2 effectors include Cas9, Cpf1, C2e1, C2e2 and C2e3. Class 2 subtypes can fall into two subclasses; those that cleave the non-target strand of the target dsDNA using a RuvC-like nuclease and those that attack RNA targets using a two HEPN domain RNase (Shmakov Nat Rv Microbiol. 2017, 15(3) 169-182).

–-Cas9 Nucleases: see also outline

Cas9 nucleases, which are components of type II CRISPR-Cas systems, are RNA guided DNA endonucleases that induce DSBs at target sites. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Type II CRISPR system from Streptococcus pyrogenes involves only a single gene encoding the Cas9 protein and two RNAs, a mature CRISPR RNA (crRNA) and a partially complementary trans-acting RNA (tracrRNA) which are necessary and sufficient for RNA guided silencng of foreign DNA). Doudna (US 10,266,850).

The Streptocococcus pyogenes SF370 type II CRISP locus consists of four genes, including the Cas9 nuclease, as well as two noncoding CRISPR RNAs (crRNAs): trans-activating crRNA (tracrRNA) and a precursor crRNA (pre-crRNA) array containing nucclease guide sequences (spacers) interspaced by identical direct repeats (DRs). Cong (Scinece, 339, 15, 2013)

The type II CRISPR enzyme Cas9, adopted from the immune system of Streptococcus pyogenes (SpCas9), is the most widely used CRISPR enzyme for genome engineering. In the interference phase, Cas9 associates with a crRNA and a short trans-activating CRISPR RNA (tracrRNA) sequence that forms a partial duplex with the crRNA. This com-plex probes double-stranded DNA (dsDNA) when it en- counters a three-nucleotide 5¢-NGG-3¢ PAM, and if there is crRNA/DNA sequence complementary, Cas9 is activated and cleaves the dsDNA between three and seven nucleotides upstream from the PAM. (Balderston, CRISP Journal, 4(3), 2021)

–Cas12 nucleases:

cas12 type V nucleases possess a single RuvC-like domain that cleaves both target and non-target strands and generates staggered ends downstream of PAM sites. Cas12a (foremly known as Cpf1) was the first Cas12 nuclease to be used as a gene editing tool. It only requires a crRNA and can self process pre-crRNA into mature crRNA, which is advantageous for multiplex gene editing. Cas12b, which reqires both a crRNA and tracdrRNA, has acheived gene editing in human cells and plants. Cas12d (formely Casy), Cas 12h and Cas12i have RNA guided DNA interference activity in E. coli. Cas12g is a RNA guided ribonuclease with collateral RNase and single strand DNase activities. Cas12e (formely CasX) and Cas12 have been adopted as gene editing tools in eukaryotic cells and Cas12f (formely Cas14) nucleases have been shown to achieve robust gene modificaiton and regulation in mammalian cells. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

–Cas13 nucleases:

Class 2 type VI CRISPR Cas systems target RNA, and the single effector nucleas belongs to the Cas13 protein family (divided into the four major subgroups Cas133a-d). Compared to other CRISPR Cas systems (e.g., the dsDNA targeting Cas9 systems, class 2 type VI systems are relatively are in bacteria. Kretz, “Function of the RNA-targeting class 2 type VI CRISPR Cas system of Rhodobacter capsulatus” Frontiers in Microbiology, 2024)

Cas13 nucleases belonging to type VI CRISPR Cas systems and contain two HEPN domains are RNA guided ribonucleases. They can process their pre-crRNA into mature crRNA, only require this crRNA to celave target RNA and have collateral activity. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

DNA Targeting CRISP

CRISPR-Cas Systems provide adaptive immunity in archaea and bacteria. In brief, the CRISPR-Cas response consists of three stages, In stage one adaptation phase, the Cas1-Cas2 protein complex excises a segment of the target DNA (known as the protospacer) and inserts it between the repeats at the 5′ end of a CRISPR array, yielding a new space. In the stage two expression and processing stage, a CRISPR array, together with the spacers, is transcribed into a long transcript known as the pre-CRISPR RNA (pre-crRNA) and is processed by a distinct complex of Cas proteins into mature small CRISPR RNAs (crRNAs). In the third interference stage, a complex of Cas proteins uses the crRNA as a guide to cleave the target DNA or RNA. (Shmakov Nat Rv Microbiol. 2017, 15(3) 169-182).

DNA targeting CRISP include Cas9 (see outline), Case12a (Cpf1), Cas12b(C2c1) and Cas12e(CasX).

CRISPR/Cas9: See outline

RNA-Targeting CRISPR

Although the primary target of the CRISPR/Cas system is DNA in most studied systems, it was shown that osme systems can target RNA. Accordingly, there is potential to leverage CRISPR loci targeting RNA to regulate or silence transcript levels within the cell. Barrangou, “CRISPR: New horizons in phage resistance and strain identification”. Annu. Rev. Food Sci, Technol 2012, 3: 143-62

RNA CRISP include Case13(a) (C2c2), Cas 13b and Cas 13d (CasRx).

RNA-guided RNA targeting CRISPR-Case factor Cas13a (previously known as C2c2):

Although some Cas enzymes target DNA, single-effector RNA guided ribonucleases (RNases), such as Cas13a can be reprogrammed with CRISPR RNAs to provide a platform for specific RNA sensing. On recognition of the RNA target, activated Cas 13a engages in “collateral” cleavage of nearly non-targeted RNAs. This collateral effect with isothermal amplifcaiton has been used to establish a CRISP-based diagnostic, providing rapid DNA or RNA detection (Gootenberg, “Nucleic acid detection with CRISPR-Cas13a/C2c2” Science e56, 438-442 (2017).

Cas13a can be engineered for mammalian cell RNA knockdown and binding. (Abudayyeh, Nature, 550, 2017).

Applications of CRISPR systems:

Most applications of CRISPR systems have focused on the programmable DNA targeting activity of Cas9. The cleavage activity of Cas9 can be harnessed for genome editing, including gene knockout and precide editing through homology-directed repair. (Shmakov Nat Rv Microbiol. 2017, 15(3) 169-182).\

Pathogen detection

Ackerman (Nature, 582, 2020) discloses using a combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN) for scalable, multiplexed pathogen detetcion. In CARMEN, nanolitre droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA target pairs on a single array and simultaneously differentiates 169 human associated viruses with at least 10 publsihed genome sequences.