Okay, if you are working in the biomedical research field and you have not heard about CRISPR technology, then you are way behind the trend curve. Don’t worry; I would like to share what I have learned about this gene-editing tool with you.

Surprisingly, this new technology came out of left field. And for the past few years, it has held the attention of the entire biomedical field.  This is not only because of its powerful gene-editing capability but also because of its accompanying ethical considerations.

In this article, I will briefly touch on the history that gave rise to the CRISPR technology. Then, I will introduce the key players in the system and how the scientists adopted this system for gene engineering work.

An Ancient Defense System

It turns out that an amazing defense system sits in the genome of many types of bacteria. CRISPR stands for “clustered regularly interspaced short palindromic repeats.” For a long time, bacteriologists realized that many bacterial genomes contain a region defined by a variety of unique sequences called “spacers” that are flanked by short inverted palindromic repeats. This finding puzzled bacteriologists at first, until someone decided to align those unique sequences to known DNA libraries.  The result they got was astounding! Can you believe many of those unique sequences matched to bacteriophage DNA sequences? That’s when researchers realized that they might be looking at an immune system bacteria use to defend against their natural predator, the bacteriophage.

Once researchers set their sight in the right direction, they were able to map out and characterize the major components of the system. So far, three different CRISPR pathways have been mapped out, and the CRISPR II system seems to be the most prominent (Fig 1). The CRISPR system establishes immunity by “remembering” components of the invading pathogen (i.e., bacteriophages) through storing the unique bacteriophage DNA sequences between the palindromic repeats. The next time the same bad guys try to invade the bacteria, the CRISPR system and a key enzyme called CRISPR-associated protein 9 (Cas9) scans the DNA pool via the use of complementary guide RNAs (gRNAs) for any intruder that matches the existing unique sequences stored in the “databank.” Cas9 then nicks the DNA and signals the DNA demolition team downstream to destroy the invader. How neat is that?!

Figure 1. CRISPR Pathways. Photo adapted from Wikimedia Commons.

Making Gene Editing Easier

Before CRISPR technology came along, scientists relied on techniques such as homologous recombination, zinc finger nucleases (ZFNs), and transcription activator like-effector nucleases (TALENs) for gene editing. Compared to the ZFNs1 and TALENs, the CRISPR system is highly specific, cost effective, and much easier to design. Another major advantage of CRISPR is the capability to “scale up.” Multiple gRNAs can be used to edit different genetic loci simultaneously, allowing you to study diseases that involve multiple genes.

Already, you can see the use of CRISPR technology to generate any gene knockouts in a very specific way. All you have to do is design a gRNA sequence,2 and co-transfect an expression plasmid containing Cas9 into a cell of your interest (that’s right, CRISPR works in many different cell types!). You are pretty much guaranteed to get the desired mutation following some screening. In addition, scientists have also modified Cas9 enzyme to enhance the specificity of the knockout region too.3

CRISPR Technology in Action

Scientists are already testing the power of CRISPR technology, as witnessed by the surge of CRISPR technology-related publications. I am just going to provide a few notable examples:

Sickle-cell anemia– a team of researchers from Boston used CRISPR technology to edit a stretch of DNA that constitutes the “enhancers” in the blood stem cells. By disabling the enhancers, scientists saw a dramatic increase in the fetal hemoglobin – a big step towards gene therapy for sickle cell diseases.

HIV– the human immunodeficiency virus (HIV) integrates into our genome and causes AIDS. Now scientists are contemplating how to use CRISPR to either inactivate or remove the integrated HIV genomes in HIV-infected patients. Already, scientists have demonstrated CRISPR-technology can remove the integrated HIV DNA in vitro. The verdict is still out to see if this technology will work in the clinical settings.4

Cystic Fibrosis- Scientists have replaced the defective CFTR gene with the wild type gene using the CRISPR system. And the functional CFTR gene restored function in vitro.5

Generation of knockout animals- Scientists now can generate knockout animals with high specificity and efficiency because both copies of the target can be removed one setting using CRISPR technology. This reduces the time it takes to generate double knockout animals, which typically requires further screening and breeding of the animals.6,7

Genetic screening for loss of functions- You can use the CRISPR system to screen for loss of function mutations using an assortment of RNA sequences (an RNA library).8

Summary of CRISPR Introduction

So, that was my brief introduction to CRISPR technology. It is hailed as the next “breakthrough” in gene editing. However, the key to its specificity lies in the designing sequence of the gRNA. Before you jump on the bandwagon, make sure you consult some references on designing gRNAs.

As we are all inching towards personalized gene editing and medicine, we should always keep in mind the kind of genetically-modified organisms we might be creating and the potential biological and ethical consequences. Otherwise, enjoy the technology and let me know what you think!

Reference

  1. Urnov FD, et.al. (2005)  Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 435: 646–51. doi:10.1038/nature03556.
  2. Fu Y, et. al. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology. 32: 279–84. doi:10.1038/nbt.2808.
  3. Mali P, et al. (2013) RNA-guided human genome engineering via Cas9Science. 339: 823–26. doi:10.1126/science.1232033.
  4. Ebina H, et. al.  (2013) Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirusScientific Reports. 3. doi:10.1038/srep02510.
  5. Schwank G, et al. (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell. 13: 653–58. doi:10.1016/j.stem.2013.11.002.
  6. Hai T, et. al. (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas systemCell Research 24: 372–75. doi:10.1038/cr.2014.11.
  7. Gratz S, et. al. (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196: 961–71. doi:10.1534/genetics.113.160713.
  8. Koike-Yusa H, et.al . (2013) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA libraryNature Biotechnology 32: 267–73. doi:10.1038/nbt.2800.