the stem-loop architecture inherent in the crispr locus, which mediates the maturation of crrna-tracrrna ribonucleoprotein complex. cas9 in complex with crispr rna (crrna) and trans-activating crrna (tracrrna) further recognizes and degrades the target dsdna. in the co-crystal structure shown here, the crrna-tracrrna complex is replaced by a chimeric single-guide rna (sgrna, in red) which has been proved to have the same function as the natural rna complex. the sgrna base paired with target ssdna is anchored by cas9 as a t-shaped architecture. this crystal structure of the dna-bound cas9 enzyme reveals distinct conformational changes in the alpha-helical lobe with respect to the nuclease lobe, as well as the location of the hnh domain. the protein consists of a recognition lobe (rec) and a nuclease lobe (nuc). all regions except the hnh form tight interactions with each other and sgrna-ssdna complex, while the hnh domain forms few contacts with the rest of the protein. in another conformation of cas9 complex observed in the crystal, the hnh domain is not visible. these structures suggest the conformational flexibility of hnh domain. to date, at least three crystal structures have been studied and published. one representing a conformation of cas9 in the apo state, and two representing cas9 in the dna bound state. interactions with sgrna crispr/cas9 in sgrna-cas9 complex, based on the crystal structure, rec1, bh and pi domains have important contacts with backbone or bases in both repeat and spacer region. several cas9 mutants including rec1 or rec2 domains deletion and residues mutations in bh have been tested. rec1 and bh related mutants show lower or none activity compared with wild type, which indicate these two domains are crucial for the sgrna recognition at repeat sequence and stabilization of the whole complex. although the interactions between spacer sequence and cas9 as well as pi domain and repeat region need further studies, the co-crystal demonstrates clear interface between cas9 and sgrna. dna cleavage cas9 nuclease and its dna cleavage position previous sequence analysis and biochemical studies have posited that cas9 contains two nuclease domains: an mcra-like hnh nuclease domain and a ruvc-like nuclease domain. these hnh and ruvc-like nuclease domains are responsible for cleavage of the complementary/target and non-complementary/non-target dna strands, respectively. despite low sequence similarity, the sequence similar to rnase h has a ruvc fold (one member of rnase h family) and the hnh region folds as t4 endo vii (one member of hnh endonuclease family). wild-type s. pyogenes cas9 requires magnesium (mg2+) cofactors for the rna-mediated dna cleavage; however, cas9 has been shown to exhibit varying levels of activity in the presence of other divalent metal ions. for instance, cas9 in the presence of manganese (mn2+) has been shown to be capable of rna-independent dna cleavage. the kinetics of dna cleavage by cas9 have been of great interest to the scientific community, as this data provides insight into the intricacies of the reaction. while the cleavage of dna by rna-bound cas9 has been shown to be relatively rapid (k ≥ 700 s−1), the release of the cleavage products is very slow (t1/2 = ln(2)/k ≈ 43-91 h), essentially rendering cas9 a single-turnover enzyme. additional studies regarding the kinetics of cas9 have shown engineered cas9 to be effective in reducing off-target effects by modifying the rate of the reaction. the cleavage efficiency of cas9 depends on numerous factors. a key requirement is the presence of a valid pam at the non-target strand 3 nucleotides downstream from the cleavage site. the canonical pam sequence for s. pyogenes cas9 is ngg, but alternative motifs are tolerated with lower cleavage activity. the most efficient alternative pam motifs for the wild-type s. pyogenes cas9 are nag and nga. the sequence composition at the target dna site complementary to the 20 nucletode spacer region of the grna also affects cleavage efficiency. the most relevant nucleotide composition properties that impact efficiency are those in the pam-proximal region. free energy changes of nucleic acids are also highly relevant in defining cleavage activity. guide rnas that bind to the dna forming a duplex that falls into a restricted range of binding free energy changes that excludes extremely weak or stable bindings generally perform efficiently. stable guide rna folding conformations can also impair cleavage. problems bacteria pose to cas9 editing most archaea and bacteria stubbornly refuse to allow a cas9 to edit their genome. this is because they can attach foreign dna, that does not affect them, into their genome. another way that these cells defy cas9 is by process of restriction modification (rm) system. when a bacteriophage enters a bacteria or archaea cell it is targeted by the rm system. the rm system then cuts the bacteriophages dna into separate pieces by restriction enzymes and uses endonucleases to further destroy the strands of dna. this poses a problem to cas9 editing because the rm system also targets the foreign genes added by the cas9 process. applications of cas9 to transcription tuning interference of transcription by dcas9 due to the unique ability of cas9 to bind to essentially any complement sequence in any genome, researchers wanted to use this enzyme to repress transcription of various genomic loci. to accomplish this, the two crucial catalytic residues of the ruvc and hnh domain can be mutated to alanine abolishing all endonuclease activity of cas9. the resulting protein coined 'dead' cas9 or 'dcas9' for short, can still tightly bind to dsdna. this catalytically inactive cas9 variant has been used for both mechanistic studies into cas9 dna interrogative binding and as a general programmable dna binding rna-protein complex. the interaction of dcas9 with target dsdna is so tight that high molarity urea protein denaturant can not fully dissociate the dcas9 rna-protein complex from dsdna target. dcas9 has been targeted with engineered single guide rnas to transcription initiation sites of any loci where dcas9 can compete with rna polymerase at promoters to halt transcription. also, dcas9 can be targeted to the coding region of loci such that inhibition of rna polymerase occurs during the elongation phase of transcription. in eukaryotes, silencing of gene expression can be extended by targeting dcas9 to enhancer sequences, where dcas9 can block assembly of transcription factors leading to silencing of specific gene expression. moreover, the guide rnas provided to dcas9 can be designed to include specific mismatches to its complementary cognate sequence that will quantitatively weaken the interaction of dcas9 for its programmed cognate sequence allowing a researcher to tune the extent of gene silencing applied to a gene of interest. this technology is similar in principle to rnai such that gene expression is being modulated at the rna level. however, the dcas9 approach has gained much traction as there exist less off-target effects and in general larger and more reproducible silencing effects through the use of dcas9 compared to rnai screens. furthermore, because the dcas9 approach to gene silencing can be quantitatively controlled, a researcher can now precisely control the extent to which a gene of interest is repressed allowing more questions about gene regulation and gene stoichiometry to be answered. beyond direct binding of dcas9 to transcriptionally sensitive positions of loci, dcas9 can be fused to a variety of modulatory protein domains to carry out a myriad of functions. recently, dcas9 has been fused to chromatin remodeling proteins (hdacs/hats) to reorganize the chromatin structure around various loci. this is important in targeting various eukaryotic genes of interest as heterochromatin structures hinder cas9 binding. furthermore, because cas9 can react to heterochromatin, it is theorized that this enzyme can be further applied to studying the chromatin structure of various loci. additionally, dcas9 has been employed in genome wide screens of gene repression. by employing large libraries of guide rnas capable of targeting thousands of genes, genome wide genetic screens using dcas9 have been conducted. another method for silencing transcription with cas9 is to directly cleave mrna products with the catalytically active cas9 enzyme. this approach is made possible by hybridizing ssdna with a pam complement sequence to ssrna allowing for a dsdna-rna pam site for cas9 binding. this technology makes available the ability to isolate endogenous rna transcripts in cells without the need to induce chemical modifications to rna or rna tagging methods. transcription activation by dcas9 fusion proteins in contrast to silencing genes, dcas9 can also be used to activate genes when fused to transcription activating factors. these factors include subunits of bacterial rna polymerase ii and traditional transcription factors in eukaryotes. recently, genome-wide screens of transcription activation have also been accomplished using dcas9 fusions named 'crispra' for activation.