2017-01-16

CRISPR/Cas 9 System-A gene editing tool

By: Mitali Padhi

What is CRISPR?

 CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.  The first description of what is today known as CRISPR was given by Osaka University researcher Yoshizumi Ishino in 1987, who accidentally cloned part of a CRISPR together with the iap gene.  The CRISPR is an array of short repeated sequences separated by spacers with unique sequences where spacers are often derived from nucleic acids of virus or plasmid. (D. Rath et al., 2015)  Each repetition is followed by short segments of “spacer DNA" from previous exposures to a bacteriophage virus or plasmid. (Marraffini and Sontheimer, 2010) 2017-01-16

Faces of CRISPR Revolution-Jennifer Doudna And Emmanuele Charpentier

CRISPR/CAS SYSTEM:

 CRISPR loci are often adjacent to cas genes (CRISPR-associated genes)  cas genes encode a large and heterogeneous family of proteins that carry functional domains typical of nucleases, helicases, polymerases, and polynucleotide-binding proteins.(Philippe Horvath and Rodolphe Barrangou, 2016)  CRISPR, in combination with Cas proteins, forms the CRISPR/Cas systems.  3 distinct types of bacterial CRISPR systems identified so far:  Type I, II, III  Type II is the basis for current genome engineering applications  From Streptococcus pyogenes  The S. pyogenes system is orthogonal to the native E. coli system 2017-01-16

CAS 9

 Cas9 (CRISPR associated protein 9) is an RNA-guided DNA- endonucleases associated with the CRISPR type II adaptive immunity system in Streptococcus pyogenes.  S. pyogenes utilizes Cas9 to interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. (Robert Heler et al., 2015)  Cas9 performs this interrogation by unwinding foreign DNA and checking whether it is 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. (Martin Jinek et al., 2012)  This is how CRISPR/Cas9 system works in bacterial adaptive immunity.

CRISPR/Cas 9 system in Bacteria

(Yingze Zhao et al,.2014) 2017-01-16

CRISPR/Cas 9

 As explained previously, CRISPR/Cas9 system is responsible for immunity of bacterial cells.  So, basically it can take in the viral genome exposed and add it in the genomic DNA.  This mechanism can be used to add desirable traits using phage vector to carry such DNA fragments.  Due to above mechanism, CRISPR/Cas9 is important tool in genetic engineering for insertion/deletion or other manipulations in DNA.

CRISPR/Cas9 Technology

(D. Rath et al., 2015) 2017-01-16

Applications:

 Gene Silencing: Using CRISPR/Cas9 technology and microRNA, gene silencing can be done so that the unwanted gene products are not obtained. (Eira Choudhary et al., 2014)  Genome editing: CRISPR/Cas9 genome editing is carried out with a Type II CRISPR system. When utilized for genome editing, this system includes Cas9, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA) along with an optional section of DNA repair template that is utilized in either Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR) (Woong Y Hwang et al., 2013)  DNA-free CRISPR/Cas9 gene editing: This system uses no CRISPR-Cas9 components in the form of DNA vectors; each component is either RNA or protein. (Je Wook Woo et al., 2015)

 Transient gene silencing or transcriptional repression (CRISPRi): With this application, Cas9 is modified so that it cannot cut DNA, and when combined with a guide RNA that targets a promoter region, the complex can reduce transcriptional activity and concomitant gene expression. (Matthew H Larson et al., 2013)  Transient activation of endogenous genes (CRISPRa or CRISPRon): By employing a Cas9 mutant that cannot cut DNA and to which a transcriptional activation domain has been fused, the expression of endogenous genes can be up-regulated by targeting the Cas9 fusion protein to the promoter region of an endogenous target gene, or multiple genes simultaneously.  Embryonic stem cell and transgenic animals: CRISPR-Cas systems can be used to rapidly and efficiently engineer one or multiple genetic changes to murine embryonic stem cells. 2017-01-16

Future Perspective and Ethical Challenges:

 CRISPR/Cas9 Technology is revolutionary tool in genetic engineering to treat genetic disorders like cancer, AIDS, Psoriasis, Diabetes, etc.  It can also be used to edit human genome and produce offsprings with desirable traits.  But in human society, it is very objectable to work on embryonic stage. There are many ethical issues which need to be addressed.  Hence, the advancement of this technique is on questionable ground due to its restricted usage.

Disadvantages

 A high frequency of off target effects has been found in human cells, but low in mice and zebrafish

 large genomes may contain multiple DNA sequences identical or highly homologous to intended target DNA sequence.

 Another important problem is the efficient safe delivery of CRISPR-Cas9 into cell types or tissues that are hard to transfect and/or infect.

 Furthermore, the disappearance of a whole population targeted by gene drive may carry drastic consequences in the ecosystem equilibrium.

 Ethical concerns have been raised regarding the possibility of genome editing in human germline, thus is the genome that can be transmitted to following generations, be from gametes, a fertilized egg or from first embryo divisions. 2017-01-16

Conclusion:

 As discussed, the CRISPR/Cas9 Technology is a very efficient, fast and accurate tool in genetic engineering.  But there are many ethical issues regarding use of this revolutionary technique.  Scientists like Jenifer Doudna spoke publically to spread awareness about it and bring world together to decide how to move forward for its enhancement.  To use it properly for the betterment of humanity and avoid misuse, few international summits were also organised to discuss future strategies.

Bibliography:

 Devashish Rath, Lina Amlinger, Archana Rath, Magnus Lundgren; The CRISPR- Cas immune system: Biology, mechanisms and applications; Biochimie 117 (2015) 119-128

 Luciano A. Marraffini and Erik J. Sontheimer ; CRISPR interference: RNA- directed adaptive immunity in bacteria and archaea; Nature Review Genetics. 2010 Mar; 11(3): 181–190.

 Philippe Horvath, Rodolphe Barrangou; CRISPR/Cas, the Immune System of Bacteria and Archaea; Science 08 Jan 2010; Vol. 327, Issue 5962, pp. 167-170

 Robert Heler, Poulami Samai,Joshua W. Modell,Catherine Weiner,Gregory W. Goldberg, David Bikard & Luciano A. Marraffini; Cas9 specifies functional viral targets during CRISPR–Cas adaptation; Nature 519, 199–202 (12 March 2015) doi:10.1038/nature14245 2017-01-16

Bibliography (cont…)

 Martin Jinek,Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier; A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity; Science 17 Aug 2012; Vol. 337, Issue 6096, pp. 816-821; DOI: 10.1126/science.1225829

 Eira Choudhary, William Bishai, Nisheeth Agarwal; Expression of a Subset of Heat Stress Induced Genes of Mycobacterium tuberculosis Is Regulated by 3',5'-Cyclic AMP; PloS one, 2014

 Woong Y Hwang,Yanfang Fu,Deepak Reyon,Morgan L Maeder,Shengdar Q Tsai,Jeffry D Sander,Randall T Peterson, J-R Joanna Yeh & J Keith Joung; Efficient genome editing in zebrafish using a CRISPR-Cas system; Nature Biotechnology 31, 227–229 (2013) doi:10.1038/nbt.2501

 Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jennifer A. Doudna, Jonathan S. Weissman, Adam P. Arkin, Wendell A. Lim; Repurposing CRISPR as an RNA- Guided Platform for Sequence-Specific Control of Gene Expression;Cell Volume 152, Issue 5, 28 February 2013, Pages 1173–1183.