Introduction to CRISPR/Cas9

Companies: Intellia Aldevron (Danaher)

Non-profits: Innovative Genomic Institute  Innovative Genomics Insttitute Education Section  Biopku   

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR assocaited) systems are prokaryotic adaptive immune systems that bind and cleave foreign nucleic acids. The most frequently used type II CRISPR system is composed of two components: Cas9 nuclease and an artificial single guide RNA (sgRNA), a fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). When the SpCas9 sgRNA complex recognizes an NGG (N=A, T, C or G) proto-spacer-adacent motif (PAM) sequence, the spacer of the sgRNA pairs with the target DNA strand to form an “R-loop” structure. Subsequently, the Cas9 nuclease cleaves the DNA strands and produces a blunt-end DSB 3 bp upstream of the PAM into the photospacer. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

The Cas9 protein (CRISPR-associated protein 9), derived form type II CRISP (clustered regularly interspaced short palindromic repeats) bacterial immune systems are an RNA guided DNA endonucleae. Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence specific gene editing much easier. The CRISPR system is an adaptive immune mechanism present in many bacteria and the majority of characterized Archaea. CRISPR containing organisms acquire DNA fragments from invading bacteriophages and plasmids before transcribing them into CRISPR RNAs to guide cleavage of invading RNA or DNA.  The Cas9 system has the potential to cure diseases. For example, when Cas9 is introduced into infected cells together with sgRNAs targets crucial viral genomic elements, it helps to inactive or clear the viral genome and thereby defends the cells from infections such as HIV, hepatits B virus, HPV and Epstein-Barr virus. (Wang, “CRISPR/Cas9 in genome editing and beyond” Annu. Rev. Biochem, 2016, 85: 22.1-22.38). 

CRISPR/Cas9 can be used for genome modification in eukaryotic cells. Guide RNAs are synthesized specific to the target location to be modified and they direct the Cas9 nuclease to make double stranded breaks. A nonhomologous end joining (NHEJ) repair mechaisms ligates the cut ends of the DNA nonspecifically, whereas a homology directed repair (HDR) mechanism repairs the double straned breaks using a donor template with homologous sequences to the cut site.  NHEJ based DNA repair is error prone and results in small nucleotide inertions and deletions that leads to subsequent frame shifts or deletion mutations and potentially to the loss of gene function. This repair pathway is used for engineering gene knockout or loss of function models. By contrast, HDR mediated repair is more accurate because it integrates a donor DNA with homology arms to the sequence at the SDB. It is thus used for site specific gene knock in and point mutation models. 

The conical Cas9 variant sources from Streptococcus pyrogenes (SpCa9) recognizes and binds to an NGG prospacer adjacent motive (PAM) and after binding cuts-3 nt upstream of the PAM, resulting in a blunt ended double strand break. Other naturally occuring Cas proteins (for exampe, SaCa9, NmCas9) offer smaller sizes and altered PAM specificites, which can be useful for viral packaging and expanding targeting ranges. Further, some Cas proteins can site-specifically target RNAs, notable Cas13 variants. In additional to repurposing naturally occurring CRISP-Cas platforms, the Cas9 protein has been engineered for imporved specificity, epxanded targeting ranges and to allow sequence modificaitons without DSBs. (Bashor, “Engineering the next generation of cell-based therpaeutics” Nature Reviews Drug Discovery)

 Zhang (US8,697,359) discloses a Type II CRISPR-Cas system having threee components: (1) a crRNA molecules called the “guide sequence”, (2) a “tracr RNA” called an “activator-RNA) and a protein called Cas9. To alter a DNA molecule, the system must acheive three interactions: (1) crRNA binding by specific base pairing to a specific sequence in the DNA of interest (“target DNA”), (2) crRNA binding by specific base pairing at another sequence to a tracr RNA and (3) tracr RNA interacting with a Cas9 protein which then cuts the target DNA at a specifi c site. The CRISPR-Cas9 system occurs naturally in bacteria (prokaryotes).

Parts to CRISPR/Cas9:

Guide (“Targetting” or “spacer”) sequence: refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided RNA binding agent. A guide sequence can be 20 base pairs in lenght such as in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs/orthologs. The guide RNA can be designed to recognize (e.g., hybridize to) a target sequence of a particular gene. 

A guide RNA for a CRISPR/Cas9 nucelase system includes a CRISPR RNA (crRNA) and a tracr RNA (tracr). For example, the crRNA can include a targeting sequence that is complementary to and hybridizes with the target sequence on the target nucleic acid molecule. The crRNA may also include a flagpole that is complementary to and hybridizes with a portion of the tracrRNA to promote the formation of a functional CRISPR/Cas complex. In some embodiments, the flagpole can include all or a portion of the sequence (also called a “tag” or “handle” of a naturally occuring crRNA that is complementary to the tracr RNA in teh same CRISPR/Cas system.  In some embodiments, the guide RNA may include two RNA molecules referred to as a “dual guide RNA”. The dgRNA may include a first RNA molecule that includes a crRNA and a second RNA molecule that includes a tracer RNA. The first and second RNA molecules may form a RNA duplex via the base pairing between the flagpole on the crRNA and the tracr RNA. In some embodiments, the guide RNA may include a single RNA molecule referred to as a “single guide RNA”. The sgRNA can include a crRNA covalently linked to a tracr RNA. In some embodiments, the crRNA and the tracr RNA may be covalently linked via a linker.  Shah (WO 2017/173054)

In some embodimetns, nucleic acid, e.g., expression cassettes, encoding the guide RNA are included. In some embodiments, the nucleic acid may be a DNA molecule.. The nucleotide sequence encoding the guide RNA may be oeprably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3′ UTR or a 5′ UTR. Shah (WO 2017/173054)

Target sequence: for Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse compliment), as nucleic acid substrate for a Cas protein is a double stranded nucleic acid. The target sequence has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent) within the target sequence. 

RNA-guided DNA binding agent (Cas Nuclease): means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence specific and depends on the sequence of the RNA. Examples of RNA guided DNA binding agents include Cas cleavases/nickases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease” also called “Cas protein” encompasses Cas cleavases, Cas nickases and dCas DNA binding agents. In some embodimetns, the RNA guided DNA binding agent is a class 2 Cas nuclease. The RNA guided DNA binding agent has cleavase activity, which can also be referred to as double stranded endonuclease activity. The RNA guided DNA binding agent includes a Cas nuclease, such as a Class 2 Cas nuclease (which may be, e.g., a Cas nuclease of Type II, V, or V1), Class 2 Cas nucleases includes for example, Cas9, Cpf1, C2c1, C2c2 and C2c3 proteins and modificaitons therof. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes. In some embodiments, the RNA guided DNA binding agent has single strand nickase activity, i.e., can cut one DNA strand to produce a single strand break, also known as a “nick”. In some embodimetns, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are compementary to the sense and antisense strands of the target sequence. In this embodiments, the guide RNAs direct the nickase to a target sequence and introduces a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, a nickase used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. A RNA guided DNA binding agent may include a nuclear localization signal (NLS). (Kanjolia US 2020/0248180). 

In S. pyogenes, Cas9 generates a blunt ended doulbe stranded break 3 bp upstream of the prtospacer-adjacent mtif (PAM) via a process mediated by two catlytic domains in the protein, an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand. (Mali, Nat Biotechno. 2013, 31(9) 833-838). Cas9 nucleases, which are components of type II CRISPR-Cas systems, are RNA guided DNA endonucleases that induce DSBs at target sites. Cas9 has two distinct nuclease domains, HNH and RuvC, which cleave the target and non-target strand respectively. Inactivaiton of either nuclease domain creates a Cas9 nickage (nCas9) which cleaves only one DNA strand. Inactivaiton of both nuclease domains generates dead Cas9 (dCas9) which still binds to garget DNA. nCas9 is useful in base editors and rpime editors, which perform precise geneome ediitng without requireing DSBs and dCas9 serves as a scaffold for recruiting effectors proximal to specific genomic sites. dCas9 is widely used for regulating transcription, altering epigenetic controls, imaging living cells and other purpsoes. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Template:  CRISPR-cas systems can also include a template nuclei acid used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some cases, the template may be used in homologous recombination which can result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In some cases, a single template may be provided but in other cases two or more templates can be provided such that homologous recombination can occur at two or more target sites. In other cases, the template may be used in homology-directed repair which involves DNA strand invasion at the site of the cleavage in the nueleic acid. In some cases, the homology directed repair can result in incluing the template sequence in the edited target nucleic acid molecule. In some caes, the template sequence may include an exogenous sequence such as a protein or RNA coding sequence operably linked to an exogenous promoter sequence such that, upon integration of the exogenous sequence into the target nucleic acid molecule, the cell is capable of expressing the protein or RNA encoded by the integrated sequence. In some examples, the exogenous sequence may provide a cDNA sequene encoding a protein or a portion of the protein. The target nucleic acid molecule may be DNA or RNA that is endogenous or exogenous to a cell. In some cases, teh target nucleic acid is an episomal DNA, a plasmid, a genomic DNA, viral genome, mtochondrial DNA or a chromosome from a cell or in the cell.

In cases involving a Cas nuclease, such as a Class 2 Cas nuclease, the target sequence may be adjacent toa protospacer adacent motif (“PAM”). In some cases, the PAM may be adjacent to or within 1-4 nucleotides of the end of the target sequene.. The PAM may be seelted from a consensus or a particular PAM sequence for a specific Cas9 protein. 

Modified CRISPR:

A group of researches have used a modified version of Cas9 bearing a D10A mutation that produces “nicks” in target DNA to produce a system they term EvolvR for dramatically increasing in vivo mutagenesis (Nature, Letter “CRISP-guided DNA polymerases enable diversification of all nucleotides in a tunable window”. This was shown in E. coli for producing cells resistant to the antibiotic streptomycin. The modified nCas9 is fused to the amino terminus a fidelity reduced variant (D424A, 1709N, A759R) of E coli DNA polyemrase I. The palsmid encoidng this contruct also encoded a guide DNA (gDNA) to target a second plasmid containing a homologous gene sequence. These expeirments shows mutations arising within a 17 nucleotide window 3′ of the nick site, consistent according to the known 15-20 nucleotide processiveity of the polymerase. Mutation rates using this system wehre further enhanced by making additional mutations in nCas-9 (K848A, K1003A, R160A); these mutations had been suggested to lower the non-specific DNA affinity of Cas-9. 

The Cas nuclease mRNA may be modified for improved stability and/or immunogenicity properties. The modificaitons may be made to one or more nucleosides within the mRNA. Examples of chemical modificaitons to mRNA ucleobases include pseudouridine, 1-mthyl-pseudouridine and 5-methyl-cytidine. The mRNA encoding a Cas nuclease may also be codon optimized for expression in a particular cell type such as a eukaryotic cell. The mRNA can also include a 5′ cap, such as m7G(5′)ppp(5′)N. The mRNA can also include at elast one element that is capable of modifying the intracellular half-life of the RNA. In some cases, teh element may be within the 3′ UTR of teh RNA. For example, the element may include a mRNA decay signal. The element may also include a polyadenylation signal.  Shah (WO 2017/173054)

Prime editors: are an alternative CRISPR-Cas based techbology that allow for the inesertion of amlls (<50 bp) sequences at a target stie. Prime editors realy on a CRISPR-Cas9 nickage, a reverse transcriptase and a modified gRNA. A Cas9 nickase is a modified Cas protein wehre one of the catalytic domains is inactivated via a mutation so that only a single DNA stran cut is made instead of cutting both strands. The modified gRNA, termed prime-editing guide RNA (pegRNA), serves two functions. First, it specifies the site for genome insrtion, and second, it provides a template for insertion into the gebome. The first IND cleared using prime editing techology is an ex vivo hematopoietic stem cell therapy wehre the prime editor is used to correct a mutation. (Christina Fuentes “Coming of age: an overview of the growing toolbox for gene editing and its use in CGT applications” Cell & Gene therapy insights, 2024; 10(9), 1221-1236). 

Thermostable Geneome Editors:

–Stearothermophilus Cas9 (GeoCas9); is stable in human serum and an efficient genome editors. In addition, by adjsuting the type of lipid used in the LNP, one can direct an RNP towrd different organs like the lungs in mice. GeoCas9 was mutated using directed evolution to optimize its ability to edit. The result was iGeoCas9 with more than 100 times the efficiency of its WT. While SpCas9 recognizes a imple NGG PAM, WT GeoCas9 requires a PAm sequence that is early 3 times as long (N4CRAA). Thus, compared to SpCas9, the editing options for GeoCas9 is much smaller. iGeoCas9, on the other hand, can recognize a elss stringent N4SNNA Pam. iGeoCas9 is more negatively charged than its peers. Thus, unlike SpCas9, it is less vulnerable to degradation by organic solvents. Thus it can potetially be delivered using LNPs whereas SpCas9 can not. The stability of iGeoCas9 based LNP-RNP complexes at 4C could significantly reduce the need for cold chain logistics –many at -70C, which are often prohibitively expensive and difficult to maintain in regions with limited infrastructure. (Hough, “Hope springs forth with therostable gebome editors” MRNA & Gene Editing, Drug Delivery, Oct 28, 2024). 

Delivery of CRISPR/Cas cargoes

Lipid Nanoparticles: 

Shah (WO 2017/173054) discloses lipid nanoparticle (LNP) based compositions for delivery of CRISPR/Cas editing components. The LPs include a plurality of lipd molecules physically assocaited with each other by intermolecular formes. The LNP compositions are preferentially taken up by liver cells (e.g., hepatocytes). The LNPs specifically bind to apolipoproteins such as apolipoprotein E (ApoE) in the blood. Apolipoproteins are prtoeins circualting in plasma that are key in regualting lipid transport. ApoE represent one class of apolipoproteins which interacts with cell surface heparin sulfate proteoglycans in th liver during the uptake of lipoprotein. The lipid compositions for delivery of CRISPR/Cas mRNA and guide RNA components to a liver cell include a CCD lip which can in some  ses be Lipd A, Lipid B, or Lipid D. The lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipids may be protonated adn thsu bear a positie charge. Covnersely, in a slightly basic medium such as blood wehre pH is about 7.35, the lipids may bear no charge. In some embodiments, the lipids may be protonated at a pH fo at elast about 9. The ability of a lipid to bear a charge is related to ints intrinsic pKa. For example, the lipids may have a pKa of from about 5l8-6.2 which may be advantageous in that cationic lipids with a pKa of about 5.1-7.4 are effective for elivery of cargo to the liver. Additional lipids can be included in the composition such as neutral lipids, uncharged or zwitterionic lipids. Helper lipids which enhance transfection of the nanoparticle can also be included. Stealth lipids that alter the lenght of time the nanoparticle can exist in vivo can also be included. In some examples, the LNPs are formted by mixing an aqueous RNA solution with an organic sovlent based lipid solution such as 100% ethanol. A buffer is sued to maintain the pH of the composition comprising LNPs for example at or aboue pH 7.0. For example, the LNPs can be formulated with a CCD lipid amine (e.g., Lipid A or Lipid B) to RNA phosphate (N:P) molar ratio of about 4.5. The lipid nanopartcile components are dissolved in 100% ethanol with CCD lipid, hleper lipid (e.g., chloesterol), neutral lipid (e.g., DSPC) nd PEG. The RNA cargo is dissolved in acetate buffer, pH 4.5 resutling in a concentraiton of RNA cargo of about 0.45 mg/ml. The LNPs are formed by micrfluidic mixing of the lipid and RNA sollutions using aPrecision Nanosystems NanoAssemblr. 

Patents

In August 2012, University of California researchers published an article (“Jinek 2012”) demonstrating that the isolated elements of the CRISPR-Cas9 system could be used in vitro in a non-cellular experimental enviornment. This led to UCs US patent Application No. 13/842,859, which relate to the use of CRISPR0-Cas9 system for the targetted cutting of DNA molecules. The system includes threee components: (1) a crRNA; (2) a tracrRNA dn (3) the Cas9 protein. The crRNA is an RNA molecule with a variable porition that tarets a particular DNA dsequence. The nucleotides that make up the variable porition complement the target sequence in the DNA and hybridize with the target DNA. Another porition of the crRNA consists of nucleotides that complement and bind to a portion of tracrRNA. The Cas9 protein interacts with the crRNA and tracrRNA and cuts both strads of DNA at the target location. (see Regents v. Broad Institute, Fed. Cir. 2018). 

Claim 165 of the application is as follows:

“A method of cleaving a nucelic acid comprising

contacting a target DNA molecule haivng a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspeaced Short Palindromic Repeats (CRISPR) – CRISPR associated (Cas) CRISPR-Cas system comprising

a) a Cas9 protein; and 

b) a single molecule DNA-targeting RNA comprising  (i) a targeting-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the tarrgeter-RNA to form a doulbe-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA dn the targeter-RNA are convalently linked to one another with intervening nucleotides, wehrein the single molecule DNA targeting RNA forms a complex with the Cas9 protein, wherey the single molecule NA-targeting RNA targets the target sequence, and the Cas9 prtoein cleaves the target DNA molecule”

In February 2013, Broad Institute researchers published an article describing the use of CRISPR-Cas9 in a human cell line, leading to US Patent No. 8,697,359. Claim 1 of the ‘359 is as follows:

“A method of alterning expression of at least one gene product comprising introducing into a eukaryotic cell containinga nd expressing a DNA molecule haivng a target sequence and encoding the gene product an engineered, non-naturally occurring clusterd regularly interspaced short palindromic repeats (CRISPR)-CRISPR assocaited (Cas) (CRISPR-Cas) system comprising one or more vectors comprising:

a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence enocding a CRISP-Cas sytem guide RNA that hybridizes with the target sequence, and 

b) a second regulatory element operable in a eukaryotic cell operaly linked to a nucleotide sequence encoding a Type-II Cas9 protein, 

wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together. “

Applications

CRISPR-engineered T cells/ Adoptive cell therapy):

The infusion of ex vivo engineered T cells, called adoptive T cell therapy, can increase the natural antitumor immune response of the patient. CRISPR-Cas9 gene editing of T cells from patients with advanced refractory cancer has already been shown effective in clinical trials. Removing the endogenous T cell receptor (TCR) (two genes encoding the TCRalpha and TCRbeta were deleted in T cells to reduce TCR mispairing and to enhance the expression of a syntehtic cancer-specific TCR transgene 9NY-ESO-1). and the immune checkpoint molecule programmed cell death protein 1 (PD-1) for example has been shown to improve the funciton and persistence of gengineered T cells. The T cell product was manufactured by electroporation of ribonucleoprotein complexes comprising recomibnant Cas9 loaded with enquimolar mixtures of single guide RNA (sgRNA) for TRAC, TRBC and PDCD1) followed by lentiviral transductino o f the trasngenic TCR. (Stadtmauer (Science 367, 1001 (2020)). 

Citrus Industry Examples:

The most devastating disease affecting the global citrus industry is Huanglonbing (HLB), caused by the pathogen Candidatus Liberibacter asiaticus. HLB is primarily spread by the inset vector Diaphorina citire (Asian Citrus Psyllid). HLB reached the Western Hemisphere by 2004 and by 2013, every grove in Florda was considered infected. To counteract the rapid spread of HLB by D. citri, tranditional cector control stragegies such as insecticide sprays, the release of natural predators and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To frutehr expand the available toods for D. citri control through geenrating specific modificaitons of the D. citir genomes, Akbari developed protocals for CRISPR-Cas9 based genetic modification. However, genome editing in D. citri has been challenging due to the general fragility and size of D. citri eggs. Akbari disclsoes methods for collecting and prparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and altenrative methods of inecting RNP into the hemocael of adult fremales for ovarian transduciton. To demonstrate genomic dNA targetting, two genes conserved in several inesct species known to produce visible phenotypic changes in eye color when disrupted, w and kh were hcoses. Mutants for tiehr gene display a reduction of pigmentation in their eyes. The genomic sequences for w and kh were identified by comparing it with the known protein sequences of the Drosophila melanogaster w and kh using tBlastn on the latest version of the D. citiri gehnome. Potential sgRNA target sites marked with a short protospacer adjacent motif (PAM, 5′-NGG-3′) across exons 1 to 6 of the w genes and on exons 7 and 8 of the kH gene were identified. Primers across both genes were then designed. The infal list of sgRNAs contained only those with high targetting efficiency and cleavage rates, resulting in three sgRNAs targeting sties on exons 2, 3 and 6 of the w genes and two sgRNAs targeting two sites on exon 7 and the kh gene. (Akban, GEN Biotechnoloy, 2(4), 2023). 

Transthyretin amyloidosis: also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. Gilmore (CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis” N Eng J. Medicine, 2021)

Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD): are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. Frangoul (“CRISPR-Cas9 gene editing for sickle cell disease and beta-Thalassemia” NEngl J Med 2021)  performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients — one with TDT and the other with SCD — received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex