cas9 cas9 cas9 (crispr associated protein 9, formerly called cas5, csn1, or csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against dna viruses and plasmids, and is heavily utilized in genetic engineering applications. its main function is to cut dna and thereby alter a cell's genome. the crispr-cas9 genome editing technique was a significant contributor to the nobel prize in chemistry in 2020 being awarded to emmanuelle charpentier and jennifer doudna. crispr-associated endonuclease cas9crystal structure of s. pyogenes cas9 in complex with sgrna and its target dna at 2.5 å resolution.identifiersorganismstreptococcus pyogenes m1symbolcas9alt. symbolsspcas9entrez901176pdb4oo8refseq (mrna)nc_002737.2refseq (prot)np_269215.1uniprotq99zw2other dataec number3.1.-.-chromosomegenomic: 0.85 - 0.86 mbsearch forstructuresswiss-modeldomainsinterpro cas9identifierssymbol?interproipr028629 more technically, cas9 is a dual rna-guided dna endonuclease enzyme associated with the clustered regularly interspaced short palindromic repeats (crispr) adaptive immune system in streptococcus pyogenes. s. pyogenes utilizes crispr to memorize and cas9 to later interrogate and cleave foreign dna, such as invading bacteriophage dna or plasmid dna. cas9 performs this interrogation by unwinding foreign dna and checking for sites complementary to the 20 nucleotide spacer region of the guide rna (grna). if the dna substrate is complementary to the guide rna, cas9 cleaves the invading dna. in this sense, the crispr-cas9 mechanism has a number of parallels with the rna interference (rnai) mechanism in eukaryotes. apart from its original function in bacterial immunity, the cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in dna. these breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. research on the development of various cas9 variants has been a promising way of overcoming the limitation of the crispr-cas9 genome editing. some examples include cas9 nickase (cas9n), a variant that induces single-stranded breaks (ssbs) or variants recognizing different pam sequences. alongside zinc finger nucleases and transcription activator-like effector nuclease (talen) proteins, cas9 is becoming a prominent tool in the field of genome editing. cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide rna. because the target specificity of cas9 stems from the guide rna:dna complementarity and not modifications to the protein itself (like talens and zinc fingers), engineering cas9 to target new dna is straightforward. versions of cas9 that bind but do not cleave cognate dna can be used to locate transcriptional activator or repressors to specific dna sequences in order to control transcriptional activation and repression. native cas9 requires a guide rna composed of two disparate rnas that associate – the crispr rna (crrna), and the trans-activating crrna (tracrrna). cas9 targeting has been simplified through the engineering of a chimeric single guide rna (chirna). scientists have suggested that cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. in 2015, cas9 was used to modify the genome of human embryos for the first time. crispr-mediated immunity to survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages, bacteria and archaea have evolved methods to evade and fend off predatory viruses. this includes the crispr system of adaptive immunity. in practice, crispr/cas systems act as self-programmable restriction enzymes. crispr loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate crispr repeats and variable crispr spacers between 24 and 48 nucleotides long. these crispr loci are usually accompanied by adjacent crispr-associated (cas) genes. in 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign dna elements, including plasmids and viruses. these reports provided the first biological evidence that crisprs might function as an immune system. cas9 has been used often as a genome-editing tool. cas9 has been used in recent developments in preventing viruses from manipulating hosts' dna. since the crispr-cas9 was developed from bacterial genome systems, it can be used to target the genetic material in viruses. the use of the enzyme cas9 can be a solution to many viral infections. cas9 possesses the ability to target specific viruses by the targeting of specific strands of the viral genetic information. more specifically the cas9 enzyme targets certain sections of the viral genome that prevents the virus from carrying out its normal function. cas9 has also been used to disrupt the detrimental strand of dna and rna that cause diseases and mutated strands of dna. cas9 has already showed promise in disrupting the effects of hiv-1. cas9 has been shown to suppress the expression of the long terminal repeats in hiv-1. when introduced into the hiv-1 genome cas9 has shown the ability to mutate strands of hiv-1. cas9 has also been used in the treatment of hepatitis b through targeting of the ends of certain of long terminal repeats in the hepatitis b viral genome. cas9 has been used to repair the mutations causing cataracts in mice. fig. 2: the stages of crispr immunity crispr-cas systems are divided into three major types (type i, type ii, and type iii) and twelve subtypes, which are based on their genetic content and structural differences. however, the core defining features of all crispr-cas systems are the cas genes and their proteins: cas1 and cas2 are universal across types and subtypes, while cas3, cas9, and cas10 are signature genes for type i, type ii, and type iii, respectively. adaptation adaptation involves recognition and integration of spacers between two adjacent repeats in the crispr locus. the "protospacer" refers to the sequence on the viral genome that corresponds to the spacer. a short stretch of conserved nucleotides exists proximal to the protospacer, which is called the protospacer adjacent motif (pam). the pam is a recognition motif that is used to acquire the dna fragment. in type ii, cas9 recognizes the pam during adaptation in order to ensure the acquisition of functional spacers. loss of spacers and even groups of several have also been observed by aranaz et al. 2004 and pourcel et al. 2007. this probably occurs through homologous recombination of the between-repeat material. crispr processing/biogenesis crispr expression includes the transcription of a primary transcript called a crispr rna (pre-crrna), which is transcribed from the crispr locus by rna polymerase. specific endoribonucleases then cleave the pre-crrnas into small crispr rnas (crrnas). interference/immunity interference involves the crrnas within a multi-protein complex called cascade, which can recognize and specifically base-pair with regions of inserting complementary foreign dna. the crrna-foreign nucleic acid complex is then cleaved, however if there are mismatches between the spacer and the target dna, or if there are mutations in the pam, then cleavage will not be initiated. in the latter scenario, the foreign dna is not targeted for attack by the cell, thus the replication of the virus proceeds and the host is not immune to viral infection. the interference stage can be mechanistically and temporally distinct from crispr acquisition and expression, yet for complete function as a defense system, all three phases must be functional. stage 1: crispr spacer integration. protospacers and protospacer-associated motifs (shown in red) are acquired at the "leader" end of a crispr array in the host dna. the crispr array is composed of spacer sequences (shown in colored boxes) flanked by repeats (black diamonds). this process requires cas1 and cas2 (and cas9 in type ii), which are encoded in the cas locus, which are usually located near the crispr array. stage 2: crispr expression. pre-crrna is transcribed starting at the leader region by the host rna polymerase and then cleaved by cas proteins into smaller crrnas containing a single spacer and a partial repeat (shown as hairpin structure with colored spacers). stage 3: crispr interference. crrna with a spacer that has strong complementarity to the incoming foreign dna begins a cleavage event (depicted with scissors), which requires cas proteins. dna cleavage interferes with viral replication and provides immunity to the host. the interference stage can be functionally and temporarily distinct from crispr acquisition and expression (depicted by white line dividing the cell). transcription deactivation using dcas9 dcas9, also referred to as endonuclease deficient cas9 can be utilized to edit gene expression when applied to the transcription binding site of the desired section of a gene. the optimal function of dcas9 is attributed to its mode of action. gene expression is inhibited when nucleotides are no longer added to the rna chain and therefore terminating elongation of that chain, and as a result affects the transcription process. this process occurs when dcas9 is mass-produced so it is able to affect the most genes at any given time via a sequence specific guide rna molecule. since dcas9 appears to down regulate gene expression, this action is amplified even more when it is used in conjunction with repressive chromatin modifier domains. the dcas9 protein has other functions outside of the regulation of gene expression. a promoter can be added to the dcas9 protein which allows them to work with each other to become efficient at beginning or stopping transcription at different sequences along a strand of dna. these two proteins are specific in where they act on a gene. this is prevalent in certain types of prokaryotes when a promoter and dcas9 align themselves together to impede the ability of elongation of polymer of nucleotides coming together to form a transcribed piece of dna. without the promoter, the dcas9 protein does not have the same effect by itself or with a gene body. when examining the effects of repression of transcription further, h3k27, an amino acid component of a histone, becomes methylated through the interaction of dcas9 and a peptide called fog1. essentially, this interaction causes gene repression on the c + n terminal section of the amino acid complex at the specific junction of the gene, and as a result, terminates transcription. dcas9 also proves to be efficient when it comes to altering certain proteins that can create diseases. when the dcas9 attaches to a form of rna called guide-rna, it prevents the proliferation of repeating codons and dna sequences that might be harmful to an organism's genome. essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dcas9 to combat the overproduction of those codons and results in the shut-down of transcription. dcas9 works synergistically with grna and directly affects the dna polymerase ii from continuing transcription. further explanation of how the dcas9 protein works can be found in their utilization of plant genomes by the regulation of gene production in plants to either increase or decrease certain characteristics. the crispr-cas9 system has the ability to either upregulate or downregulate genes. the dcas9 proteins are a component of the crispr-cas9 system and these proteins can repress certain areas of a plant gene. this happens when dcas9 binds to repressor domains, and in the case of the plants, deactivation of a regulatory gene such as atcstf64, does occur. bacteria are another focus of the usage of dcas9 proteins as well. since eukaryotes have a larger dna makeup and genome; the much smaller bacteria are easy to manipulate. as a result, eukaryotes use dcas9 to inhibit rna polymerase from continuing the process of transcription of genetic material. structural and biochemical studies crystal structure crystal structure of crispr-associated protein cas9, based on pdb 5axw by nishimasu et al. cas9 features a bi-lobed architecture with the guide rna nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange, and gray). these two lobes are connected through a single bridge helix. there are two nuclease domains located in the multi-domain nuclease lobe, the ruvc (gray) which cleaves the non-target dna strand, and the hnh nuclease domain (cyan) that cleaves the target strand of dna. the ruvc domain is encoded by sequentially disparate sites that interact in the tertiary structure to form the ruvc cleavage domain (see right figure). crystal structure of cas9 in the apo form. structural rendition was performed using ucsf chimera software. a key feature of the target dna is that it must contain a protospacer adjacent motif (pam) consisting of the three-nucleotide sequence- ngg. this pam is recognized by the pam-interacting domain (pi domain, orange) located near the c-terminal end of cas9. cas9 undergoes distinct conformational changes between the apo, guide rna bound, and guide rna:dna bound states. cas9 recognizes