Biotech World: January 2016

Saturday, January 30, 2016

CRISPR and genome editing

CRISPRs or Clustered Regularly Interspaced Short Palindromic Repeats are short prokaryotic DNA sequences (~ 20 nt) that are part of bacterial adaptive immune system. Recent research in genomics suggests that these CRISPR could change/edit the DNA sequence at exact location on a chromosome of almost any type of living being, including humans, very fast. CRISPR gene editing technology is much easier, cheaper and faster than other gene editing methods including Zn fingers and TALENs (Transcription Activator Like Effector Nucleases). There are 3 types of CRISPR mechanisms among which type II is the most studied one. The key components of CRISPR gene editing are Cas9 protein / Csn1 endonuclease along with the guide RNA (crRNA and trRNA) [1]. In CRISPR pathway first the foreign DNA is integrated into the CRISPR locus and the loci are then transcribed to produce crRNA. These crRNA then guide the Cas9 / RNAse III family endonuclease to edit the target genome or to destroy the invading DNA in sequence specific way [2].

This CRISPER or Cas9 based genetic technology has various applications in biotechnology and medical research for gene knock out, gene repression/activation, genetic screening, genomic loci imaging and purification [3], epigenetic modifications, transcriptional regulation, etc. This powerful gene editing method has huge potential to treat genetic disorders and cancer, to produce genetically modified crops, to engineer the ecosystems through gene drive, to produce transgenic animal model for biomedical research, in drug development and most recently (in April 2015) to edit human embryonic stem cells (though it triggered ethical debate). In 2014 researchers from MIT first used CRISPER editing in mice to treat a metabolic disease tyrosinaemia. In January 2016, scientists from Duke University successfully used CRISPER genetic technology to treat DMD (Duchenne Muscular Dystrophy, a genetic disorder that causes muscle breakdown due to mutation in dystrophin gene) in mouse model [4]. As Cas9 could correct the causative mutation this CRISPER/Cas9 genome editing technology may direct an exciting future in therapeutics to treat monogenic recessive disorders/genetic diseases [5]. Examples of such disorders include sickle cell anaemia, cystic fibrosis, DMD, retinitis etc. Though scientists need to overcome many hurdles this Cas9 based genome editing technology has powerful future solution for effective genome modification that lead to novel inventions in biomedical and genomic research.

References:

1. https://www.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology

2. http://www.nature.com/nature/journal/v482/n7385/fig_tab/nature10886_F1.html

3.CRISPR/Cas9 Guide: https://www.addgene.org/CRISPR/guide/

3.http://www.sciencealert.com/crispr-gene-editing-tool-used-to-treat-genetic-disease-in-an-animal-for-the-first-time

5. Patrick D. Hsu, Eric S. Lander, Feng Zhang, Development and Applications of CRISPR-Cas9 for Genome Engineering, DOI: http://dx.doi.org/10.1016/j.cell.2014.05.010

Friday, January 15, 2016

RNA silencing in plant biotechnology

RNA silencing or post transcriptional gene silencing (PTGS) plays an important biological role in plants which includes the following:

defense against viruses
epigenetic control of chromatin modifications
control of the expression of developmentally regulated genes
regulation of biotic & abiotic stress, and
defense against nonviral pathogens and insects.

RNA silencing pathways or RNA interference (RNAi) are mediated by small homologous RNA molecules that are 21 to 24 nt long. These RNA molecules are processed by dicers or dicer-like endonucleases (DCL) and RNA dependent RNA polymerases (RDRs). They bind with AGO (Argonaute) proteins to form RISC (RNA induced silencing complex) and repress/modify the gene expression at transcriptional, post transcriptional and translational level. RNA silencing pathways involve various types of small RNAs which includes

micro RNAs (mi RNAs) that have important biological role in plants including auxin regulator and accumulator of transcription factors that involves in plant development [1]
endogenous trans acting si RNAs/TAS (ta-siRNAs)
natural cis antisense transcripts associated si RNAs (nat si RNAs), and
heterochromatic si RNAs (hc si RNA).

The micro RNAs are produced by DCL1 and binds with AGO1 to cleave target mRNA and repress the translation. The ta-si RNA is produced by DCL4 and RDR6 to cleave TAS gene and control the gene expression in plants [2]. The hc si RNAs are produced by plant specific transcript POL4, RDR2 and DCL3 and then binds with AGO4, AGO6 and AGO9 to direct DNA methylation. Through these RNA silencing pathways plants are defended against invaded viral nucleic acid/transgenes. During this defense mechanism viral RNA is diced by DCL4&DCL2 to produce the si RNAs to degrade the viral RNA [3].

RNAi is now widely used in industrial plant biotechnology for production of disease and pathogen resistant plants, for generation of male sterility to produce hybrid seeds, for crop production with improved nutritional contents and in various other aspects. Industrially RNAi has also been used to modify the metabolic pathways of plants to enhance nutrient accumulation and decrease toxin generation. For example, lyc gene and DET1 gene in tomato are engineered by RNAi to increase lycopene (antioxidant), flavonoid and beta carotene concentration. FAD2 and SAD1 gene in canola, cotton, and peanut plant is manipulated by RNAi to increase the oleic acid and stearic acid concentration [4]. In 2015 a group of scientists at Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO) explained the use of RNAi to increase the healthier monounsaturated fatty acid (MUFA) level in flaxseeds [5]. CSIRO used RNAi to reduce the function of 3 genes in cottonseed that usually convert oleic acid to bad fatty acids. This resulted in high oleic acid containing cottonseed oil that is free from cholesterol raising trans fatty acids [6].

Other than plants RNAi technology is also applied for genome analysis and drug target validation for therapeutic development and hence it is termed as "breakthrough in biotech industry".