CRISPR

The podcast episode discusses the use of CRISPR technology to differentiate stem cells into bone and cartilage cells, with a focus on cartilage repair. The current methods for joint cartilage deterioration involve artificial replacements, but the goal is to use stem cells and CRISPR technology to create chondroblasts, which can be injected into the joints for repair. CRISPR-Cas9 is a gene editing tool that allows for precise changes to the DNA sequence. The process involves using an RNA guide to find and cut the DNA in the desired location, and then the stem cell's repair machinery is used to introduce the necessary nucleotides for the desired outcome. This technique shows potential for creating many differentiated chondroblasts quickly. The article also discusses the delivery methods and possible drawbacks of this approach. Good afternoon. Welcome to the podcast RecomboNation. I'm Rick Rogers, and I'm here with Kim Ruchel and Aidan Holt, both nursing majors, with leading research and information on the science of using CRISPR to differentiate undifferentiated stem cells into bone and cartilage cells. Today's focus will be only on stem cell differentiation into cartilage cells, or those cells that exist within our joints, such as the hip, the knee, and disc of the spine. The title of the article is Genetic Scissors, CRISPR-Cas9, Genome Editing, Cutting-Edge CRISPR Technology for Bone and Cartilage Repair. First, a little background information on the state of technology and joint cartilage deterioration. Physicians replace joints of the hip and the knee and the disc of the spine if they deteriorate. Replacements are either artificial, as is the case with the hip and the knee, or artificial or bone, as is the case of the disc of the spine, called spinal fusions. In 2021, there were 2.2 million hip and knee replacements and 1.62 million spinal fusions for a total of 3.82 million cartilage procedures performed. While each procedure has been used to benefit many patients, our thought is that we will one day look back on these days as days of a bygone era, with undue pain to patients, as well as side effects, infections, and enormous cost to each individual's annual income in the way of insurance throughout several of the Western nations. The technologies have advanced in many ways during the last 10 years, and there are two in particular. One is the growth, maintenance, and usage of stem cells in humans, and in another realm, but simultaneously being developed, is CRISPR for gene modification. Stem cells are cells that exist, and they are the master cells of all cells until differentiation and maturity occur, at which point a heart cell is a heart cell, and a brain cell is a brain cell, and so on. And stem cell injections have been used in human joints without complications, but with minimal effectiveness in knee and hip joints. So how do we program the stem cell to become the cell we want them to be, in this case, a chondroblast or young new cartilage cell? This is where CRISPR comes into play. CRISPR, or the acronym stands for Clustered Regularly Interspaced Short Palindromic Repeat, was discovered in E. coli in 1987, and the version of CRISPR outlined here, CRISPR-Cas9, was invented in 2008. For several years, science has been refining CRISPR to be used safely for a variety of potential applications. In short, CRISPR-Cas9 is a strong gene editing tool, which allows for a precise nucleotide sequence to be cut out of the gene and replaced in favor of a more applicable complement of nucleotides. This is where stem cell and CRISPR-Cas9 research intersect. The use case we are examining for this article is safely using CRISPR-Cas9 on a stem cell or mesenchymal cell to produce a chondroblast. The mesenchymal cells are obtained from the patient's bone cells, and they are equivalent to the master cell that we discussed earlier for the cells of the cartilage as well as the bone. Then, what we do is we differentiate them into chondroblasts, and we inject them into the knee space or the hip space for an ideal repair of the joint. The same would also apply to spinal disc if necessary. So how do we make this happen? CRISPR-Cas9 is made up of two different components, an RNA guide called gRNA and Cas9, an enzyme that cuts DNA. The guide RNA is only about 20 nucleotides. It finds the stem cells' complementary DNA base pairs and binds to the target DNA sequence, and there are no other sequences that match the complexity of possible DNA sequence permutations. The Cas9 follows the gRNA and cuts both strands of DNA in the precise location required. The stem cell recognizes the damage to the DNA and tries to repair it using several methods in its repair machinery. Scientists use the cell's repair machinery to introduce the nucleotides to produce the expected outcome. In other words, the DNA is reprogrammed to be the desired sequence to allow the mesenchymal cell to form the chondroblast. Next, conceptually, many differentiated chondroblasts could be created with this technique in a very short period of time, or cell cultures could be used to develop mini chondroblasts. The cells are then ready for the patient. Hayden is going to take it from here and cover the delivery methods outlined in the article, and Kim is going to draw conclusions about the article itself, the science, and possible drawbacks.