Complimenting CRISPR: Improving Genetic Delivery Techniques with Nanoparticles

Christina Cai and Erin Parlow


            Christina Cai is a rising high school junior. She will be attending the Texas Academy of Math and Science in the fall where she will continue to expand upon her passion for biology and other STEM courses. Although college applications are still a year away she wishes to enter a university majoring in biochemical engineering. In her free time she enjoys dancing, reading and traveling with friends and family.

Erin Parlow is a rising high school senior. She attends Cardinal Mooney Catholic High School in Marine City, Michigan. She is very excited about biology, particularly nanobiomaterials and genetics. She will soon begin the college application process and hopes to secure a spot at an accredited university where she will study biomedical engineering and material science. In her free time, Erin enjoys playing soccer, doing community service, and working with her robotics team.



The recent sequencing of the human genome, and the consequential knowledge about the genetic code that defines humanity imparts novel capabilities in medicine and genetic engineering. Using biological cues, scientists can engineer an organism’s genome and constitute significant changes to cell function. However, despite the major strides that have been made in genetic understanding, the ability to feasibly make genetic changes still remains out of reach. This is in part due to the lack of efficient delivery systems to accompany genetic technology.8,31,32,37 As systems for genetic editing have been perfected, an increasing amount of attention is being delegated to developing delivery systems to accompany them. No matter how precise editing technology becomes, worth can only be measured in the accuracy, specificity, and lessened toxicity with which a system is introduced to target cells and their nuclei. In order for the potential of editing technology to be maximized, a delivery mechanism that effectively addresses these concerns is essential to further progress.



New understanding of the CRISPR Cas-9 immunity system has led to the discovery of a more sophisticated technique for genetic editing. The CRISPR system is a major component of the immune systems of several species of bacteria and archaea.4,15,20,25By incorporating pieces of foreign RNA into its own loci, CRISPR is able to recognize and attack identical sequences of pathogenic DNA that invade bacteria.20 CRISPR can include RNA from virtually any organism.25 Upon introduction to a cell of the same organism, CRISPR is able to use RNA to target the matching DNA sequence within the cell’s genome (Figure 1).  CRISPR splices host DNA and introduces new genes or silences the expression of genes that are already present.25 One of the earliest demonstrations of CRISPR altering the genetic material of organisms was demonstrated with cell exposure to the bacterium Streptococcus thermophilus (S. thermophilus).9 In this study, RNA spacer molecules that code for immunity to different phages were derived from various mutant versions of S. thermophilus. These were incorporated into CRISPR systems and inserted into the genome of cells, which resulted in added immunity to each of the phages.9  CRISPR machinery can transpose genes from multiple sources, inducing minimal toxicity and mutagenic activity.15 As impressive as the CRISPR vision is, it still suffers from the fundamental limitation of genetic editing. This is the absence of a proper delivery technique to transfer prepared editing systems to the targeted gene.15,25

Traditionally, cellular delivery of CRISPR has been facilitated by viral or retroviral vectors. These systems make use of biological viruses that have been genetically altered to be non-pathogenic.12 Despite these changes, there are still significant instances of viral immune response, inflammation, tissue degradation, and overall toxicity to the targeted cell.21,38 These hindrances are key motivations for the development of new delivery systems that do not rely on the infectious properties of viruses and instead make use of other nonviral particles, particularly those that are defined on the nanoscale.


Nanoparticles and LNPs

Nanoparticles are synthetic or naturally occurring molecules that measure 1-100 nanometers in at least one dimension.32,34,27 Studies have shown that with pertinence to genetic editing, the size of the particle for delivery is relevant, with nanoscale design allowing for the development of many different particles with a varied range of specifications.26  Within the nanoscale range, there are nuances in optimal particle size. Larger particles are unable to efficiently diffuse through the membrane of the cell and deliver their product.10 Conversely, particles that are too small are unable to encapsulate required machinery.30

Among nanoparticles, the most commonly studied for potential gene editing transportation are lipid nanoparticles (LNPs). Occupying an optimal size range of 10-100 nm, LNPs are formulated by synthesis of a phospholipid bilayer with positively charged lipid molecules.18,29 These capsules mimic the physical and chemical makeup of the cell membrane, allowing LNPs to more readily be taken into the cell interior.29 Once inside the cell, LNPs protect genetic machinery until successful delivery into the nucleus. One study reported that a certain LNP-mediated delivery resulted in 90% success rate of genetic recombination within the inner mouse ear.33 Success of this magnitude could suggest that nonviral nanoparticle delivery of CRISPR might be able to overtake that of traditional viral methods.


Material Composition

Although LNPs form the basis of nanoparticle delivery systems, other materials have been tested to determine whether other alternative nanoparticles exist. Tests have shown that increased efficiency can be achieved through the use of gold nanoparticles rather than LNPs in certain situations.17 Gold nanoparticles with less than a 10 nm diameter are able to easily and efficiently enter the nucleus of a breast cancer cell to delivery the gene editing system.17 Virus-Like Particles (VLPs) are nanoparticle protein structures derived from bacteriophages.27 These structures behave like viruses with lessened possibility of initiating an immune response.27 It has been shown that the use of VLPs can increase the efficiency of CRISPR targeting due to ability to resist degradation in tissue fluid.27 The fact that different compositions of nanoparticles yield varying delivery results suggests that success is dependant on the material that is being used for delivery.


Toxicity and Limitations

Despite the positive implications that result from nanoparticle delivery systems, there are still detrimental effects that can occur. Toxicity, the measure of a material’s ability to cause cell death,  is still a concern with nanoparticle systems. LNPs are able to cause lethal toxicity in laboratory mice at certain dosages.24 Toxicity has also been shown to result from nanoparticle delivery systems that are based off the polymer polyethlylenimine (PEI).28 This polymer alone has fatal effects on cell activity, although in vitro tests have shown that when combined with DNA, these toxic effects are reduced.28 After DNA is released from PEI, toxicity returns and has adverse effects on the cell. Several other polymers and synthetic lipid carriers follow this model of toxicity.28 Other instances of nanoparticle toxicity result from their interference with enzymes that promote critical metabolic processes.24 The interference of foreign material with enzymes (enzymatic inhibition) is also a form of toxicity.24 The incidence of cell toxicity caused by nanoparticle delivery systems is a point of further research to improve efficiency and biocompatibility. This alone stands as the final barrier between the successful implementation of nanoparticles as the ideal genetic editing delivery mechanism.



Perfecting genetic editing could lead to clinic incorporation of genetic editing techniques, but is currently limited by the absence of an efficient delivery system. Nanoparticles provide promising evidence for a solution to the lack of non-toxic, consistent, and efficient delivery system. Nanoparticles of various sizes and material specifications are demonstrating an increased ability to deliver their products to ideal locations. As suitable nanoparticles are for gene delivery systems, there are still challenges that need to be addressed in order to maximize the potential of this technology. Increasing the efficiency of vector transport and abolishing any toxicity that might be related are such examples. Recent proof of concept for genetic editing with CRISPR opens the doors to the vast possibilities of genetic engineering. All that remains is the discovery of a partner to accompany and facilitate delivery. Recent trends indicate that this position might soon be occupied by a nanoparticle


The Future

In the future, there will be an ideal (size, material, charge) genetic delivery system that evades the foreign body reaction, prevents inflammation, and allows for targeted delivery to cells. This will give humans the ability to consistently make changes to organisms with techniques that operate in vivo. Genetic engineering has the possibility to eradicate previously terminal genetic diseases, differentiate stem cells, or correct genetic mutations in cancerous tumors. There is an unlimited possibility that begins only with us.          



            We would like to express the most sincere gratitude to those that have allowed this blog article to come to fruition. Special thanks to our Teacher’s Assistants, Danielle Nikal and Chia-Ying Lee, for their dedication and guidance throughout the entire process of blog writing. Finally, thanks to our Stanford Pre-Collegiate Summer Institutes Professor Dr. Jagannath Padmanabhan for inspiring us to think creatively and critically about science, and for challenging us to learn and accurately communicate the field of science.



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