BE308 CRISPR_Prashant.pdf

CRISPR/CAS9 A GENE EDITING TOOL By: Prashant Agrahari [email protected]  Introduction In the late 1800s, Ernest Hanbury Hankins reported that water from the Ganges and Yamuna rivers in...

CRISPR/CAS9 A GENE EDITING TOOL By: Prashant Agrahari [email protected]  Introduction In the late 1800s, Ernest Hanbury Hankins reported that water from the Ganges and Yamuna rivers in India contained an antibacterial agent that killed Vibrio cholerae. These filterable agents, later termed as bacteriophages. CRISPR is a natural bacterial defense system against invading viruses and nucleic acids. Over billions of years, multiple CRISPR-type immune systems have evolved. Naturally occurring CRISPR systems are typically classified on the basis of CRISPR-associated (cas) genes, which are often found adjacent to the CRISPR arrays. The DNA-editing capacity of CRSPR-Cas9 is due to the ability of the WT Cas9 protein to cause double-stranded breaks at the target site that is determined by the custom-designed short guiding RNA (sgRNA). The repair of DNA breaks frequently results in indels, due to the non-homologous end joining (NHEJ) repair mechanism. However, when a complementary template is available, homology-directed repair (HDR) machinery can use it and thereby achieve more precise gene editing.  CRISPR LOCUS (Cas Operon) Locus means the physical location of a gene or a DNA segment on a chromosome. Cas9 enzyme unwinds and cuts double stranded DNA, resulting in the degradation of large stretches of DNA. CRISPR RNA is a noncoding RNA containing the spacer sequence and repeat element. The role of Cr RNA is to guide CrRNA:tracrRNA:Cas9 complex to the bacteriophage DNA. Single guide RNA (g-RNA) : A guide RNA generated by the covalent linkage of the crRNA to tracrRNA. Trans-activating CRISPR RNA, or tracrRNA, is also a noncoding RNA that hybridizes to the repeat sequence in the crRNA and is required for protospacer targeting. It binds to CrRNA and guides is to Cas9 and formed effector complex. Mechanism & Classification of different CRISPR/ Cas system 1.Adaptation 2.Expression 3.Interference Different Cas Proteins & their functions  Mechanism of CRISPR/Cas 9 system Adaptation Adaptation has two main steps: the acquisition of new spacers and the integration of spacers into the genome. This stage is directed by the nearly universal Cas1–Cas2 proteins. Acquisition occurs when foreign DNA enters the cell and is interrogated by Cas. The system copies a small piece of the foreign DNA and incorporates it into the host’s genome. DNA from an invading source ( Bacteriophage) is termed a protospacer, whereas incorporated DNA is termed a spacer. Spacers are added iteratively, with new spacers consistently being added at the leader end of the repeat–spacer array.  Expression The next stage is expression or biogenesis of CRISPR RNA (crRNA). Transcription begins at the leader end of the repeat–spacer array.  Interference The process of targeting nucleic acids is termed interference. This stage is directed by the effector complex. The effector complex ( CrRNA:trCrRNA:Cas9) is formed, recognizes foreign nucleic acids, and generates nucleic acid cleavage. DNA is scanned for a PAM site.Cas9 binds to the non complementary strand of the PAM and then checks for sequence similarity between the spacer in the crRNA and the target DNA. If no mismatches are identified in the seed region, then Cas9 enacts an exact double-strand break through the use of two nickase. Protospacer adjacent motif (PAM): short nucleotide sequence found adjacent to the protospacer on the nontarget strand of the nucleic acid. The PAM sequence is recognized by the Cas9 protein and initiates nucleic acid targeting  DNA Repair mechanism Homology-directed repair (HDR): template-dependent DNA repair mechanism results in the error free repair of a break or insertion of a novel DNA sequence at the break. Insertion and/or deletion (indel): outcome of the NHEJ pathway creating insertion or deletion of base pairs at break site.  The DNA damage can be repaired by NHEJ yielding short random insertions or deletions at the target site. Alternatively, a DNA sequence that shows partial complementarity to the target site can be inserted during HDR for precise genome editing purposes.  Mutations in the catalytical domains of Cas9 yield a dead variant (dCas9) that binds but does not cleave DNA. The approach with dCas9 is used for transcriptional repression by binding to the promoter region of a gene and thus blocking the access for the RNA polymerase. Similarly, dCas9 can be fused to a transcriptional repressor. Red crosses represent inhibition of transcription. The fusion of dCas9 to a transcriptional activator stimulates transcription of an adjacent gene by recruiting the RNA polymerase.  CRISPR APPLICATIONS Knockouts (KOs): Loss of gene functions by the INDELs( Insertion or deletion) are introduced in the genome. Knock-ins(KIs): Gain of gene functions by the INDELs are introduced in the genome. nCas9(Nickase Cas 9): Modified cas 9 protein to cleave one of the two strand of the target DNA. Application of CRISPR/Cas in therapeutics Application of CRISPR/Cas in the food industry  Ethical concerns about CRISPR technology The ethical concerns about CRISPR genome engineering technology are largely due to at least three important reasons. Firstly, there are concerns about the power and technical limitations of CRISPR technology: These include the possibilities of limited on-target editing efficiency, incomplete editing, and inaccurate on- or off-target editing. These limitations have been reported in CRISPR experiments involving animals and human cell lines. As more efficient and sensitive CRISPR tools are developed, many of these concerns may become outdated. The second concern is about the future of the modified organisms: There are worries about whether the changes will last forever and if the edited genes will be passed to future generations, possibly causing unexpected issues. Uncertainty resulting from these factors hinders accurate risk/benefit analysis, complicating moral decision making. The Doubtful perspective is that even if genome editing works as planned and produces the desired results, we still don’t fully understand how genetic changes affect biological traits. As a result, the impact of editing genes in germline or somatic cells might be uncertain and vary depending on the situation.