November 27, 2020

The Niche

Knoepfler lab stem cell blog

CRISPR critters advancing science & agriculture

CRISPR applications in the real world

Mina Kim, The Niche Blog
Author Mina Kim, intern on The Niche Blog.

The “breakthrough” gene-editing tool, CRISPR Cas9, has been utilized for various purposes since its popularization and commercialization in the early 2010s.

While possible uses of gene editing in humans tend to get the most attention, the application of CRISPR-Cas9 also encompasses the animal world and the analysis of a multitude of species from common livestock to exotic, wild animals.

The use of CRISPR gene-editing in these animals involves constructing animal models for the research of disease mechanisms and enhancing specific genes to improve agricultural productivity in both their and our world. 

Over the years, gene-edited animals collectively have started being called by umbrella terms like CRISPR critters and CRISPR-y critters.

CRISPR-edited animals in China

With an extremely competitive research and development budget from the government, China has been a leading country in employing CRISPR genome editing techniques mainly for diseases and agriculture research. A broad range of animals — mice, rats, pigs, dogs — have been subject to gene-editing research with a variety of goals including higher quality meats, human organ transplants, disease treatments, and healthier livestock, though they are mostly still only ideas. While often difficult to perform in other countries due to cost and access, CRISPR editing in monkeys has also been intensively studied in China in recent years. 

In 2014, Ji, Niu, and colleagues were the first to genetically edit monkeys using CRISPR. Since then, they have capitalized on CRISPR’s efficiency to create multiple monkey models for diseases like muscular dystrophy, autism, and cancer. Presently, China has at least four active research groups working on monkey gene-editing at a large scale. Recently, at the Yunnan Key Laboratory, Ji and Niu’s team harvested six rhesus macaque eggs, with, unfortunately, only one egg being developed enough to fertilize. The embryo that is mature enough to fertilize will then be injected with one rhesus sperm and, if successful, edited with CRISPR to carry a mutation that causes exceptionally fast aging

CRISPR monkeys
CRISPR’d monkeys. “After using CRISPR to edit a gene that disrupts circadian rhythms in a monkey, Chinese researchers then produced five clones”.

CRISPR-edited Livestock

For the increase in the demand for animal-based food as well as for the production of higher-quality, productive traits, scientists have been implementing CRISPR in livestock animals. Though gene-editing in larger animals raises issues of time, money, and ease, a growing human population requires high-demands for food, one of the major challenges to our future. 

One area of focus in livestock genome editing is the attempt to decrease disease susceptibility. An outbreak in a population of livestock can be detrimental to the animals themselves of course, meat production, and public health. To combat this potential harm, CRISPR has been used to create a generation of disease-resistant animals. For example, in pigs, a new generation of pigs was created by gene editing to resist Porcine Respiratory and Reproductive Syndrome Virus (PRRSV)—the cause of the pig industry’s major economic losses. PRRSV entry into host cells is mediated by CD163, a macrophage surface protein. Thus, CRISPR came into the picture, disrupting the CD163 gene and leading to the resistance of PRRSV infection in the pigs. 

Another application of CRISPR in livestock is the enhancement of productive traits. Cashmere goats have been edited in China to improve the production of meat and the goats’ distinctive high-quality hair. Researchers developed a Cas9 mRNA and sgRNA mixture that targets the goats’ MSTN (muscle development) and FGF5 (hair length) genes, and after injection into one-cell embryos a 26.5% efficiency  was achieved. 64 pregnancies ensued, and, from 98 individuals (with 93 delivered and 5 aborted) that were genotyped, 26 lambs showed a disruption in either MSTN, FGF5, or both genes.   

An agricultural biotech firm collaborated with UC Davis researchers to make gene-edited cattle lacking horns. Usually, in horned cattle, the horns are removed when the animals are young as those that don’t have horns display less aggressive and harmful behavior, but it is a labor-intensive and painful process for the cattle. For this reason, making hornless cattle could be highly beneficial. While hornless cattle were successfully generated, in the gene-editing process it appears a segment of bacterial DNA was inserted into the bovine genome. As a result, it is unlikely that meat from the cattle could get needed governmental approval. 

Looking to the future, the team also now has promising results on a method to generate more male offspring as male cattle are 15% more productive at turning feed into weight gain. For this process, the researchers inserted the SRY gene, a gene that initiates male development, into a bovine embryo and targeted at the bovine chromosome 17, a genetic safe harbor site. With the SRY gene knock-in, the bull is expected to produce 75% male offspring.

CRISPR'd goats
“The tousled look is in for cashmere goats” — X. Wang. New Scientist.

CRISPR-edited exotic animals

More recently, researchers have been delving into the use of CRISPR on more exotic animals—animals that have never been tested or even analyzed in the labs. The variety of animals being researched with CRISPR allows the expansion of model organisms, yet, until recently in 2016 when the US National Science Foundation initiated a $24 million program for this research, there were little contributions to both cost-efficient and timely production of animal models. 

Tessa Montague at Columbia University studies the Hawaiian bobtail squid (Euprymna scolopes) and the dwarf cuttlefish (Sepia bandensis), both of which visibly portray brain activity through camouflage. In other words, the two species display what they see on their own skin. Despite the prevailing mystery and difficulty in analyzing the connection between brain activity and behavior, Montague and her team were the first to successfully inject CRISPR machinery into the embryos of the Hawaiian bobtail squid and dwarf cuttlefish and are potentially using this method to modify their neurons to light up as they fire

Hawaiian bobtail squid (Euprymna scolopes)
Hawaiian bobtail squid (Euprymna scolopes) “alters the camouflage patterns on its skin based on what it sees.” Nature.

Challenges 

As game-changing as the integration of CRISPR use in live organisms is, the recent applications of CRISPR in exotic animals pose several obstacles, considering that these animals have not yet been thoroughly analyzed in the lab. There are fundamental challenges of breeding and maintaining exotic species as well as determining the optimal method of CRISPR injection into their embryos. Not many of the more unusual organisms have had their entire genomes sequenced either, which presents another challenge as for gene editing it is necessary to know specific genomic sequences.

Despite these sometimes difficult hurdles, increasing the boundaries and specificity of model organisms for research has proven to be vital in our understanding of many species’ genomes, behaviors, and life cycles