Question

Background:

Being able to accurately identify specific genes on bacterial, plant and animal chromosomes and then modify them, has been one of the recent goals of molecular biology. Over the years various endonuclease systems, such as zinc finger nucleases, have been devised and used. However, their targeting ability was limited and many “off target” effects have happened.

In 2012, a new term erupted in the biological literature – CRISPR, which is an acronym for Clustered Randomly Inserted Short Palindromic Repeats. In the short time since then, CRISPR use has mushroomed and could easily become the dominant methodology in the future.

Your Task:

The focus of this challenge is for you to research CRISPR, explain how it works, and then explain how it can be used to edit genomes of bacteria, plants and animals. In the case of humans, technically whatever we can do in animals, can be done in humans. This raises significant ethical issues, e.g. are we playing GOD. Explore this minefield and try to arrive at some conclusions about whether we should do this to the human genome or not.

• How does CRISPR work?

• Information on gene gene knock-outs (where a functional gene can be shut down or eliminated)

• Information on gene knock-ins (where a function can be inserted or restored, or single or multiple nucleotide replacements can be used to repair a gene function)

• How can it be used to edit genomes of bacteria, plants, and animals?

• What are your conclusions about whether CRISPR should be used in humans? Are there cases where you feel it should be used? Are there cases where you feel it should not be used? Where would you draw the line?

Answer 1

1. CRISPR is a technology that can be used to edit genes by by removing, adding or altering sections of the DNA sequence.  CRISPR-Cas9 system consists of two key molecules that introduce mutation into the DNA. A) An enzyme called Cas9 that acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. B). A piece of RNA called guide RNA (gRNA) which   consists of about 20 bases long pre-designed RNA sequenc located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. The guide RNA will only bind to the target sequence and no other regions of the genome. The enzyme Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA. When the cut is repaired, mutations are introduced that usually disable a gene.

2. CRISPR is already widely used for scientific research in many of the microbes, plants and animals and may have been altered with CRISPR. In humans CRISPR system conferes resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. CRISPR is used in genome editing is of great interest in the prevention and treatment of a wide variety of diseases, including single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection. In bacteriat the CRISPR system protects prokaryotic cells by destroying foreign DNA after it has entered the cell. In plants CRISPR is used to knock out genes, CRISPR–Cas nucleases can be used to target coding regions or regulatory elements. In plants CRISPR is also used to improve germplasm particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.