DeHORN with CARE - Ethical Biotechnology

INTRODUCTION

Biotechnology is a multidisciplinary field that involves using living organisms, cells, and biological systems to develop products and technologies for various applications. It combines principles from biology, chemistry, engineering, and computer science to manipulate biological systems, for medicinal, agricultural, industrial or environmental processes. 

Humans strive for continuous development and improvement as they slowly start decoding the secrets of the universe. The history of evolution repeatedly showcases the need to exhibit extreme caution while humans toy around with the secrets of universe, till they gain sufficient proficiency and wisdom to prudently use these human induced modifications. 

As we embrace these innovations, it's crucial to approach them with a mindset of caution. There are multiple issues which arise from these techniques, such as ethical issues and the requirement of regulatory frameworks which prevent the misuse of these technologies. Perhaps the most prominent implication is the fact that despite its low chance of occurrence, unintended changes to the targeted genome can occur and it will probably not manifest itself, physically or mentally, until several generations later. 

This blog puts the latter in focus while exploring an interesting story of a Minnesota based bioinnovation center, Recombinetics Inc. and their quest to revolutionize the farming industry. 

GENE EDITING - An Overview

Recombinetics Inc. is famous for its gene editing techniques, such as CRISPR-Cas9 or TALENs. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary gene editing technique, which centers around the presence of repeated sequences of nucleotides in the genome of most prokaryotes, such as bacteria. 

☝For ease of understanding, it can be considered as writing a computer program that performs regularly repeated actions using "looping statements" rather than repeating the same statements multiple times to achieve the same functionality. This method, makes use of an enzyme, Cas9 (CRISPR-associated protein 9), to target those repeated sequences and make changes to them.

To edit these sequences more efficiently, a guide RNA molecule was used, much like Clippy, the guide and assistant in older Microsoft systems, that allows for the precise targeting of weak spots in a code and makes it more robust. The combination of the Cas9 protein
along with the guide RNA would break both DNA strands of the bacteria, which would then repair the otherwise lethal cuts, using their own mechanisms (such as Homology Directed Repair; this was basically a copy-paste of a given desired template into the existing code through the breaks).

These repeated sequences were observed in atleast 50% of all prokaryotes and 90% of all archaea (the first unicellular organisms on Earth), which meant this technique could be used for a wide range of organisms, hence increasing its importance. This has been used to identify cancer-causing genes and is undergoing trials as a potential cure for HIV. 

TALENs is similar, however this is performed directly inside the living organism, using Transcription Activator-Like Effector Nucleases (these enzymes are meant to mimic restrictive enzymes used in the cutting of DNA fragments).

Before all of this however, even now, there was the method of transfer of a required gene, using a ‘vector’, which is an artificially created or natural vehicle for the transport of the new gene to the cells of other organisms. The most common is a plasmid, a circular, free-floating DNA found mainly in prokaryotes. 

☝One can consider vector to be like a female 'Anopheles' mosquito that spreads the malaria microbe. It can also be considered as a computer virus on a USB drive which contains a particular edit to a source code. Inserting this USB drive ‘implants’ the edit into the source code, hence completely changing it. In a similar way, the plasmid would ‘implant’ that modified gene into the existing genome, thus changing it. 

Map of a plasmid used in the project

These technologies opened up many opportunities, allowing Man to alter the script of Nature, by changing the genome, which was created by Nature over millions of years of evolution.

 

PIONEERING THE ADVENT OF HORNLESS CATTLE

It was quite a revelation when, in 2016, Recombinetics announced the birth of the first hornless cattle, Buri and Spotigy, two Holstein dairy cows that had been subjected to these gene editing techniques, mainly TALENs. 

Hornless Holstein cow

This was complemented by the use of ‘Corynebacterium glutamicum’ bacteria (found in human gut and soil) and their plasmid vectors (☝remember this; it will be important later on), to edit 200 base pairs (the nitrogenous bases of DNA; “steps” of the ladder) of the gene, during the embryonic stage of these cattle, that coded for the formation of keratin required for the growth of horns from the skull (called “POLLED”). 

But what is the use of hornless cattle?

Well, cattle herding is common across the world and part of the technique involves cutting their horns periodically, to reduce risk of injury to both humans and other animals, and to ensure their easier upkeep. These horns are tough, made from closely packed keratin, and deep-rooted in the skull, hence cutting them would also cause a great deal of pain to the poor cattle. 

But what if we eliminated that step, by eliminating the root cause for that step? 

We do have the material required to give rise to genetically modified breeds that do not grow horns in their lifetime. And it made a lot of sense as well, from an economical and ‘care-for-animals’ point of view. There was a high level of anticipation and funding for this company, as a result of this revelation. 

This was indeed promising, and it was quite the leap forward in the commercial applications of gene editing. It was shown as a 'highly precise and targeted' process, with almost zero percent chance of foreign materials being left behind, hence they were highly confident and proposed that checks from the FDA, the Food and Drug Administration, were not even required.

But come 2019, the world would realize that even if there is a negligible chance of a potential impact, it must never be ignored. One needs to be absolutely sure and needs to verify every aspect, leaving no room for speculation.

 

2019 :: A FLAW IN THE PRECISE PROCEDURE GONE

In 2019, a “routine” check was conducted by the FDA (The Food and Drug Administration; they set the guidelines for producing food products and medicines, in the USA), on the cows, for they were going to be used for production of food products, such as milk, beef etc., just like any other cow farmed. They examined the data related to the edited genome and, along with crew from the company, were in for a huge shock. 

Alongside the edited gene, they found a plasmid insertion in it. They later found out that the plasmid was from, you guessed it, Corynebacterium glutamicum. The very same bacterium used in the ‘TALENs’ procedure, as a transport for the enzymes and the modified gene.

This plasmid should not have been there. Every plasmid used is designed to be transient i.e. it completes the job of transferring and replacing a given sequence with the edited gene, and leaves. However, it integrated itself into the bovine genome (the genetic sequence found in the given cattle). 

How exactly did it happen?

In this case, one cannot say for sure, however we could assume that the “POLLED” gene shares some common sequences with the bacterial plasmid, which means that the enzymes, used to make cuts at these sequences, would actually form “sticky ends” (these are open nucleotide bases formed after cleavage of a DNA segment; they pair with their complementary bases on the other sticky end and enable the use of the same). 

Two antibiotic resistance sequences, one for ampicillin resistance (AmpR, as shown in the plasmid map under 'Techniques of Gene Editing') and the other for kanamycin (KanR; both broad-spectrum drugs), also of bacterial origin, were found as well. This meant that this particular edited sequence could survive the effects of the given antibiotics, hence it could spread easily.

But this surely meant one thing. The company had not conducted their mandatory checks properly, citing a ‘very low chance of integration of foreign DNA in the cattle’ and that the sequences were targeted with very high precision. 

FDA regulations mandate that “all bioengineered products, like food, medicine etc., must not contain any kind of foreign DNA, harmless or not, and that the required safety evaluations must be conducted thoroughly”. These checks ensure that the standards of bio-engineered products meet those of the natural ones. It did not matter that the bacterium used was non-pathogenic and harmless. Such rules exist, to convey to the people that bio-engineered products will be just as safe as the normal products, like how we convince people that one drug for a certain disease is just as safe as the existing one, but more effective.  

However, in this instance, something was missed .. was it a real case of overconfidence - just because a procedure has a one in a million chance of failing does not mean it will never fail .. or was there some lapse in the verification suggested by the FDA regulations .. one can never tell.

CONSEQUENCES

This discovery had two immediate and one long-term consequence. 

We know about the ethical issues that arose in the field of biotechnology, ever since the Human Genome Project of 1990-2003. The presence of foreign material in the cattle raised doubts about the safety of the product upon consumption, due to the possible presence of unwanted toxins or byproducts. After all, the foreign DNA could re-integrate itself at anytime. 

Think about it, all the tools required for the gene-editing process, with no one to control it; the nuclease used here for the TALENs procedure, or the “Cas” protein can be recycled or degraded. This cannot be controlled without external interference and, hence, there always was a small chance of it influencing any unintended gene, like the one that produces milk or affects the immunity of the cattle, which could cause life-threatening consequences to the cattle.

With that being said, we cannot leave out the fact that animals cannot ask consent in our understood language, hence we may actually be doing such activities by violating the rights of these living beings, hence breaching the basic ethical code i.e. consent from any living being must be obtained before any test, be it a blood test, drug test or a potentially life-changing test.

Now, we know that these animals failed the FDA test. This became news worldwide, and every single country rejected the offer of housing these bio-engineered cattle. With that, the main aim of creating these cattle and propagating them across the world was crushed. The animals were nothing more than a ticking time bomb; it was only a matter of time before the bacterial DNA would spread through their offspring. Hence, for ‘protective’ purposes, they incinerated the cattle (On a side note, “Spotigy” was already killed before this, to analyse his tissues, while “Buri” sired 17 offspring, most of whom have been euthanized, with some spared for further research). So much for ‘painless’ procedures that the cattle had to undergo. 

Now with all of these losses, both of consumer trust and the FDA tests, they lost credibility, due to which the company funding reduced significantly. 

CONCLUSION - WITH GREAT POWERS COMES GREAT RESPONSIBILITIES

(Borrowing the above quotation from Spider-Man movie) 

This case reminds us that big strides in the field of biotechnology carries both immense potential and significant responsibilities. 

While the ambition to create hornless cattle aimed at improving animal welfare and reducing farming challenges, the unexpected integration of foreign DNA raised profound ethical, health, and regulatory concerns, while underscoring the critical need for transparency, rigorous safety assessments, and ongoing dialogue among scientists, regulators, and the public.

As for the company, they are slowly recovering .. a tough lesson learnt but one that would ensure safe practises in biotechnology and gene editing. Much like every aviation accident which lead to innovations and corrections for a safer air travel.

With that, I take your leave, dear reader! And I shall see you in the next one!

!!! ADIOS AMIGO  !!!