CRISPR Editing for AgBio

Agriculture Livestock Biofuel Pest Control RNP Solutions

Application Solutions » CRISPR Gene Editing » CRISPR Editing for AgBio

Overview

CRISPR gene editing is currently applied in crops and livestock, biofuel production, and invasive species or pest control to revolutionize agricultural biotechnology (AgBio).

Many studies utilize non-viral CRISPR ribonucleoprotein (RNP) systems to safely engineer more resilient or productive strains. CRISPR guide RNA and Cas nuclease protein are complexed as RNPs before being co-delivered to the cell.

Ex vivo non-viral CRISPR ribonucleoprotein (RNP) systems offer a safe, efficient, and inexpensive gene therapy alternative. In these systems, CRISPR guide RNA and Cas nuclease protein are complexed before being co-delivered via electroporation or lipofection into hematopoietic stem and progenitor cells (HSPCs), which are then reinjected into the patient.

RNP-based CRISPR systems offer several advantages over traditional plasmid-based methods, including no exogenous DNA/transgene integration, minimized off-target editing, reduced toxicity, lower manufacturing cost, and the ability to titrate dosage.

Here we examine how researchers apply transgene-free CRISPR RNP gene editing to address global problems such as climate change, food and water security, and disease.

CRISPR Applications

Agriculture

CRISPR can accelerate crop improvement compared to traditional selective breeding methods. RNP systems are preferred for crop editing, as the introduction of transgenes can cause strains to be subject to genetically modified organism (GMO) regulations.

Researchers have delivered RNPs into staple crops to knockout susceptibility genes for improved disease resistance^1,2 and to knock in single-stranded DNA (ssDNA) templates containing mutations for improved herbicide resistance³. Other CRISPR RNP studies have focused on optimizing crops for increased yield^4,5, improved nutritional profiles⁶, drought⁷ or salinity tolerance⁸, and many other desirable traits.

The predominant RNP delivery methods for gene editing in plants include polyethylene glycol (PEG)-mediated cell transfection⁶ and biolistics⁹; however, recent studies have successfully demonstrated electroporation¹⁰ and lipofection¹¹ delivery in protoplasts. Editing efficiencies vary significantly depending on plant species, RNP delivery methods, and sample types.

Livestock

CRISPR gene editing is also being applied to improve livestock breeds. In addition to the benefits listed previously, RNP-based systems are especially beneficial for time-sensitive editing in animal zygotes/embryos¹², as CRISPR RNP complexes begin editing immediately upon delivery.

CRISPR RNPs have been used to knockout myostatin in pig embryos¹³ for improved muscle mass and to knock in ssDNA templates containing mutations in bovine zygotes¹⁴.

The predominant RNP delivery method for gene editing in animal embryos is intracytoplasmic microinjection¹⁵; however, recent studies have demonstrated reliable and efficient results from electroporation¹⁶ and lipofection¹⁷ delivery.

Biofuel

In addition to agricultural crops and livestock, CRISPR is being used to optimize the production of “green energy” biofuels derived from microalgae.

Studies have demonstrated successful electroporation of CRISPR RNPs to knockout genes in a variety of microalgae species^18,19,20, and to knock-in ssDNA templates²¹ containing mutations. While these studies are currently proof-of-concept, researchers aim to use CRISPR editing to improve lipid production, growth rates, and physiological tolerances.

Invasive Species/Pest Control

Genome editing also offers the ability to control invasive species populations and pest species that cause disease to humans, crops, and livestock.

CRISPR RNPs have been used to edit genomes of malaria-vector mosquito species^22,23, as well as invasive agricultural pests silverleaf whitefly²⁴, fruitfly²⁵, and spider mite²⁶, two species of livestock-infesting flies²⁷, and the grain-pest red flour beetle²⁸.

Delivery of CRISPR RNPs into oviparous species can be achieved by embryonic injection, however this method is costly and inefficient. Recent studies have demonstrated more efficient delivery with “direct parental” injection into adult female ovaries²⁹, which can also be applied to viviparous species, such as aphids and flies.