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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).
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'''
==== 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 amount of 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.<ref>'''Jensen ''et al. Microb Cell Fact (2017) Transcriptional reprogramming in yeast'''''
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 amount of 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.<ref name="pmid28298224">{{cite journal | vauthors = Jensen ED, Ferreira R, Jakočiūnas T, Arsovska D, Zhang J, Ding L, Smith JD, David F, Nielsen J, Jensen MK, Keasling JD | display-authors = 6 | title = Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies | journal = Microbial Cell Factories | volume = 16 | issue = 1 | pages = 46 | date = March 2017 | pmid = 28298224 | pmc = 5353793 | doi = 10.1186/s12934-017-0664-2 | url = }}</ref> 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 transcripted piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body.<ref name="pmid29056323">{{cite journal | vauthors = Pinto BS, Saxena T, Oliveira R, Méndez-Gómez HR, Cleary JD, Denes LT, McConnell O, Arboleda J, Xia G, Swanson MS, Wang ET | title = Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9 | journal = Molecular Cell | volume = 68 | issue = 3 | pages = 479–490.e5 | date = November 2017 | pmid = 29056323 | pmc = 6013302 | doi = 10.1016/j.molcel.2017.09.033 }}</ref>


⚫
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.<ref name="pmid28973434">{{cite journal | vauthors = O'Geen H, Ren C, Nicolet CM, Perez AA, Halmai J, Le VM, Mackay JP, Farnham PJ, Segal DJ | title = dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression | journal = Nucleic Acids Research | volume = 45 | issue = 17 | pages = 9901–9916 | date = September 2017 | pmid = 28973434 | pmc = 5622328 | doi = 10.1093/nar/gkx578 }}</ref>
'''''using dCas9 and combinatorial gRNA strategies 16:46'''''

'''''DOI 10.1186/s12934-017-0664-2'''''</ref> 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 transcripted piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body. <ref>Pinto, S. Belinda et al. '''''Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9 Molecular Cell 68, 479-490 November 2, 2017, 2017 Elsevier Inc.'''''</ref>

⚫
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.<ref>O’Green Henriette et al. '''''dCas9-based epigenome editing suggests acquisition'''''

'''''of histone methylation is not sufficient for target gene'''''

'''''Repression Nucleic Acids Research, 2017, Vol. 45, No. 17 9901–9916'''''</ref>


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.
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.<ref>'''''Lowder L.G., Paul J.W., Qi Y. (2017) Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems. In: Kaufmann K., Mueller-Roeber B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY'''''</ref>
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.<ref name="pmid28623586">{{cite journal | vauthors = Lowder LG, Paul JW, Qi Y | title = Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems | journal = Methods in Molecular Biology (Clifton, N.J.) | volume = 1629 | issue = | pages = 167–184 | date = 2017 | pmid = 28623586 | doi = 10.1007/978-1-4939-7125-1_12 }}</ref>


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.<ref>'''''Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017).'''''</ref>
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.<ref name="pmid28581505">{{cite journal | vauthors = Barrangou R, Horvath P | title = A decade of discovery: CRISPR functions and applications | journal = Nature Microbiology | volume = 2 | issue = | pages = 17092 | date = June 2017 | pmid = 28581505 | doi = 10.1038/nmicrobiol.2017.92 }}</ref>


=== Genomic editing in eukaryotic cells ===
=== Genomic editing in eukaryotic cells ===

Revision as of 06:25, 18 November 2018

CRISPR-associated endonuclease Cas9
Crystal structure of S pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A ˚ resolution.[1]
Identifiers
OrganismStreptococcus pyogenes M1
Symbolcas9
Alt. symbolsSpyCas9
Entrez901176
PDB4OO8
RefSeq (mRNA)NC_002737.2
RefSeq (Prot)NP_269215.1
UniProtQ99ZW2
Other data
EC number3.1.-.-
ChromosomeGenomic: 0.85 - 0.86 Mb
Search for
StructuresSwiss-model
DomainsInterPro

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to memorize[2] and later interrogate and cleave foreign DNA,[3] such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 basepair spacer region of the guide RNA. 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. Alongside zinc finger nucleases and 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.[3] 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.[4] Versions of Cas9 that bind but do not cleave cognate DNA can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression.[5][6] While native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide – the CRISPR RNA (crRNA), and the trans-activating RNA (tracrRNA).,[3] 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.[7] In 2015, Cas9 was used to modify the genome of human embryos for the first time.[8]

CRISPR-mediated immunity

Introduction

To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages, bacteria and archea have evolved methods to evade and fend off predatory viruses. This includes the CRISPR system of adaptive immunity. In practice, CRISPR acts as a self-programmable restriction enzyme. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24-48 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.[9] 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.[10][11] 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.[12] In addition, Cas9 has already been used in human trials in the treatment of cystic fibrosis and oncogenic mutations in human pluripotent stem cells.[13] Cas9 has used to repair the mutations causing cataracts in mice.

Overview of CRISPR-Cas types and function

Fig. 2: The Stages of CRISPR immunity
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.

CRISPR-Cas defense stages

  1. 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.[14] In type II, Cas9 recognizes the PAM during adaptation in order to ensure the acquisition of functional spacers.[2]
  2. CRISPR Processing – 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).[15]
  3. 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.[16]

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[2]), 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 amount of 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.[17] 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 transcripted piece of DNA. Without the promoter, the dCas9 protein does not have the same effect by itself or with a gene body.[18]

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.[19]

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.[20]

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.[21]

Genomic editing in eukaryotic cells

While genomic editing in eukaryotic cells has been possible using various methods since the 1980's the methods employed had proved to be inefficient and impractical to implement on a larger scale. Genomic editing leads to irreversible changes to the gene. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via Homology Directed Repair, is the traditional pathway of targeted genomic editing approaches.[22] This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template.[22] This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for a repair to commence. Knock-out mutations caused by Cas9/CRISPR results in the repair of the double-strand break by means of NHEJ (Non-Homologous End Joining). NHEJ can often result random deletions or insertions at the repair site disrupting or altering gene functionality. Therefore, genomic engineering by CRISPR/Cas9 gives researches the ability to generated targeted random gene disruption. Because of this, the precision of genomic editing is a great concern. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. Cas9 allows for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRna guide strands.[23] Cas9 derived from S. pyogenes has facilitated the targeted genomic modification in eukaryotic cells. The ease by which researches can insert Cas9 and template RNA in order to silence or cause point mutations on specific loci has proved invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. New engineered variant of the Cas9 nuclease have been developed that significantly reduce off-target manipulation. Called spCas9-HF1 (Streptococcus pyogenes Cas9 High Fidelity 1), has a success rate of modification in vivo of 85% and undetectable off-target manipulations as measured by genome wide break capture and targeted sequencing methods used to measure total genomic changes.[24][25]

Cellular modeling

Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Cas9 can be easily introduced into the target cells via plasmid transfection along with sgRNA in order to model the spread of diseases and the cell's response and defense to infection.[26] The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function, mutation effects, all while avoiding the of off-target mutations typically observed with older methods of genetic engineering. The CRISPR and Cas9 revolution in genomic modeling doesn’t only extent to mammals. Traditional genomic models such as Drosophila Melanogaster, one of the first model species, have seen further refinement in their resolution with the use of Cas9.[26] Cas9 uses cell-specific promoters allowing the a controlled use of the Cas9. Cas9 is an accurate method of treating diseases due to the targeting of the Cas9 enzyme only affecting certain cell types. The cells undergoing the Cas9 therapy can also be removed and reintroduced to provide amplified effects of the therapy.[9]

Structural studies of Cas9

Overview

Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue; Fig. 2) 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 Figure 3).

Fig. 3: Crystal structure of Cas9 in the Apo form, as solved by M Jinek et al. in their 2014 Science paper. 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, which are detailed below.

Crystal structures in detail

Cas9 recognizes the stem-loop architecture inherent in the CRISPR locus, which mediates the maturation of crRNA-tracrRNA ribonucleoprotein complex.[27] Cas9 in complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) further recognizes and degrades the target dsDNA.[28] In the co-crystal structure shown here (Fig. 4), 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.[3] 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). It should be noted that 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.

So far, 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.

  1. Jinek et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, Feb 2014 [29]
  2. Anders et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature Sept 2014 [30]
  3. Nishimasu et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell Feb 2014 [1]

The interactions between sgRNA and Cas9

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.[1][30] 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.

Target digestion

Previous sequence analysis and biochemical studies have suggested Cas9 contain RNase H and HNH endonuclease homologous domains which are responsible for cleavages of two target DNA strands, respectively. These results are finally proved in the structure. Although the 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). Previous works on Cas9 have demonstrated that HNH domain is responsible for complementary sequence cleavage of target DNA and RuvC is responsible for the non-complementary sequence.[31]

Problems Bacteria Pose to Cas-9 Editing

Most Archea and Bacteria stubbornly refuse to allow a cas-9 to edit their genome. This is due to the fact that they are able to attach foreign DNA, that does not affect them, into their genome. Another way that these cells defy cas-9 is by process of “Restriction Modification System”. When a bacteriophage, a virus that infects bacteria and archaea and uses them to replicate itself, enters a bacteria or archea 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 cas-9 editing because the RM system also targets the foreign genes added by the cas-9 process.[32]

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.[33] 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.[34] Also, dCas9 can be targeted to the coding region of loci such that inhibition of RNA Polymerase occurs during the elongation phase of transcription.[34] In Eukaryotes, silencing of gene expression can be extented by targeting dCas9 to enhancer sequences, where dCas9 can block assembly of transcription factors leading to silencing of specific gene expression.[6] 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.[34] 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.[35] 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.[34] This is an 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.[34] 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.[36]

Another method for silencing transcription with Cas9 is to directly cleave mRNA products with the catalytically active Cas9 enzyme.[37] 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.[34] 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.[36]

Possible Solutions to Bacterium Cas-9 Editing

Recent studies have shown that with some minor modification to the cas-9 protein it is possible to bypass the “Restriction Modification System”. The process that achieves this is called “Photospacing” which means that the cas-9 system will copy a section of DNA from the host and insert it as a spacer gene into its own genome.[38]. Though we do not fully understand why “photospacers” work we do understand what they do. When a prokaryote “senses” a part of its own genetic coding inside the foreign entity it is less likely to destroy the cas-9 insertion. This is known as the beginning of cas-9 immunity.[39]

Impact Of Human Intervention

On Archean

With the discovery of the natural cas-9 style editing in bacteria and archaea humans have been learning over the years to use this system to restore or change improperly functioning genomes. While the basis for using cas-9 in eukaryotic organisms is further along we are now attempting to learn more about using this same system to repair and change genomes in prokaryotes. Though cas-9 additions are repelled by the RM system in prokaryotes destroying any foreign DNA placed by cas-9 we are learning how to work around this system and the implications are incredible. With the ability to change the genome of harmful bacteria humans could open an entirely new field of medicine and save more lives than ever before.[39]

On Bacterium

Archaea are not harmful to humans as they do not carry viruses that can hurt us, we are still interested in the possibility of editing archaeon genes. One sub-species of archaea in particular could play a vital role in our planet’s future, Methanogenic Archaea. Methanogenic Archaea are part of the carbon cycle of our planet. They use the Carbon Dioxide (CO2) in the air and combine it with Hydrogen (H2) to create Methane (CH4) and Water (H2O)[40] If the process could be sped up it, by way of genomic editing, it could vastly reduce the amount of Carbon Dioxide in our atmosphere. While there is not much know about gene editing in archaea, we do know that archaea are slow growing cells and recent data shows that it does seem to be easier to edit archaeon genomes then bacterial.

Cas9, Pros and Cos

Pros

In the world there are a lot of different things that can help or hurt plant life, ocean life and life in the air. Cas9 or CRISPER has pros and cos of helping the ecosystem in whole, and especially the coral life, but the coral life has a huge impact on all see life, some organisms more than others. Not only does CRISPER can help plant life and ocean life it can help humans directly.  There are new studies coming out that CRIPSER can help treat cancer by modifying T-cells to help find and destroy cancer cells, Not only can it help the fight against cancer, it can take out the genes that cause diabetes, and other genetic disease. This only works though if scientist have located what gene the disease is located on. One huge player in the pro column is that it can help crops against pest resilience, this is a huge player because this allows plants not to die allowing more food for us or maybe it's for fuel like ethanol. Lastly another pro for CRISPER is that it’s a pretty cheap technology so drug research and discovery could go by fast. This technology could help benefit human life as we know it, help eliminate disease, not only that It could help humans it could help every life form plant or animal. This could help with hunger, nutrient and growth of all types of creatures.[41][42][43]

Cons

Cas9 has had controversial backlash. Cas9 therapy involves the reworking the functions of the cells. It has been hypothesized that the Cas9 enzyme will disrupt or alter the functions of neighboring cells. Cas9 has also been shown to target viruses that are outside the specified parameters due to certain similarities in the viruses. Cas9 can tolerate a number of mismatches in the genomic sequence; allowing the Cas9 enzyme to split parts if the genomic sequence that falls outside the target sequence. In addition, Cas9 is feared to change the genetic makeup of future generations if it is allowed to be used in humans for the therapy. Many people have skepticism towards the Cas9 therapy in the humans due to the fear of unknown arbitrary effects in the human genome in future generations.

Are you playing GOD, what point do you say genetic modify is okay and not, so the ethical concerns because a big factor. Next thing it could lead to bioweapon, weapons that could use bacteria or virus that are not like any other and that do not have a cure. This is a major play because if terrorist organization ever got a hold of this technology it could be bad for the whole world. Another thing that could go bad with CRISPER that raises some eyebrows is that it can cause some safety issues, since this a fairly new technology some concerns are what could be some side effects. Even though we might fix one genetic problem but could that cause some other underlying problem that could possibly be life threatening.  Germline cell and embryo genome editing has a lot of ethical problems, how will we dictate this technology on weather a person is using it for medical reasons or just to help the physical makeup like their height or just their hair or eye color. So figuring out how we are going to regulate and change this is a major factor, because this might cost money and cause problems in the medical field. Also this is currently illegal for many countries, so it will have to go through the judicial system and legislator. This could cost money to the government and could be tied up in the courts for a while so when would the date we would actually see it come to in effect.[44][45]

Case Study Through Coral Reefs

Background: Coral reefs are some of the most important organisms in the sea. They provide a habitat for much marine life, assist in carbon and nitrogen fixing and protect coastlines from damaging effects of tropical storms. These reefs are being threatened by a phenomenon called coral bleaching which is caused by global warming. Once the water is too warm, coral are “bleached,” and become white.

Problem: Coral reefs are susceptible to bleaching from global warming and pollution. When water in the reefs gets too warm, coral expel algae and lose their color. Although this doesn’t cause the coral to die, the bleaching puts the coral under stress, and makes them unattractive to symbiotic organisms. This takes away “investors” in the coral, and causes the coral do die from lack of nutrients provided by the other organisms. Coral can recover from bleaching, but only if they have not starved. Once starved, symbionts are unrecoverable, and the likelihood of recovery is reduced. However, even if symbionts are recovered, reproductivity capacity is reduced leading to long term damage in coral reefs.

Solution: In order to make coral more resistant to bleaching, CRISPR Cas9 is being implemented. The long-term goal is to alter genes in the coral, making them more resistant. However, the project geneticists are working on now is a database- one that will hold all information on coral genes. This way, experts will know all they need to know before genetically engineering the coral. Once the database has been created, the plan is to permeate coral embryos using CRISPR-Cas9 and implement the genetic changes needed for resistance to bleaching. Once this is achievable, geneticists want to use CRISPR-Cas9 on existing (adult) coral cells. [46][47][48][49][50]

See also

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Further reading

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