“The power to control our species’ genetic future is awesome and terrifying. Deciding how to handle it may be the biggest challenge we have ever faced.”
― Jennifer A. Doudna
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is a component of bacterial immune systems that can cut DNA, and has been repurposed as a gene editing tool. It acts as a precise pair of molecular scissors that can cut a target DNA sequence, directed by a customizable guide.
The system is made up of two key parts: a CRISPR-associated (Cas) nuclease, which binds and cuts DNA, and a guide RNA sequence (gRNA), which directs the Cas nuclease to its target. It was discovered in bacterial immune systems, where it cuts the DNA of invading viruses, called bacteriophage, and disables them. Once the molecular mechanism for its DNA-cleaving ability was discovered, it was quickly developed as a tool for editing genomes.
CRISPR is important because it allows scientists to rewrite the genetic code in almost any organism.
It is simpler, cheaper, and more precise than previous gene editing techniques. Moreover, it has a range of real-world applications, including curing genetic disease and creating drought-resistant crops.
Today, CRISPR is well-known as a precise gene editing tool, but it took many years for scientists to figure out what it was and how to harness its potential. Let’s discuss the inventors of this gene editing tool, the scientists who championed this technology, and the history behind these breakthroughs.
It wasn’t until 2020 – well after it had been adopted in labs around the world – that Doudna and Charpentier won the Nobel Prize in Chemistry for their discovery, becoming the first all-female team to do so.
Other significant contributors include Feng Zhang at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, who pioneered the use of CRISPR in eukaryotic cells and discovered novel Cas variants, George Church at Harvard Medical School in Boston, Massachusetts, who was among the first to demonstrate its use in human cells, and biochemist Virginijus Siksnys at Vilnius University in Lithuania, who independently discovered the ability of CRISPR to edit genes in other organisms. For more information on these early pioneers, you can check out our blog about prominent CRISPR scientists.
While Doudna and Charpentier were the first to adapt CRISPR-Cas9 as a gene editing tool, the history goes back a little further than their 2012 publication. In 1993, Dr. Fransisco Mojica, a scientist at the University of Alicante in Spain, identified repetitive palindromic segments of DNA interspaced with other fragments of genetic material in bacterial genomes. Dr. Mojica gave these regions the name CRISPR, and proposed that they are a component of the bacterial immune system. In 2007, a team of scientists led by Dr. Philippe Horvath experimentally demonstrated Mojica’s theory.
Since its adaptation by Dr. Doudna and Dr. Charpentier, this versatile gene editing technology has progressed rapidly. It has been adapted for many different purposes, including RNA editing, base and prime editing, live imaging, and diagnostics. It has been used to edit DNA in a variety of organisms, including humans.
In 2019, the first CRISPR clinical trials began, harvesting cells from patients with sickle cell disease (SCD) and editing them in vitro before infusing them back into the body - a method known as cell therapy. After the success of SCD cell therapy trials, a CRISPR treatment was injected directly into human patients for the first time in 2020. This technique is known as gene therapy, and was used to treat hereditary blindness.
Want to know how to optimize your CRISPR experiment to get guaranteed editing results? Schedule a free discovery call and project consultation with a genome engineering expert.
The CRISPR system is the basis of adaptive immunity in bacteria and archaea. It utilizes Cas nucleases, which are enzymes that can bind and create double-stranded breaks (DSBs) in DNA. When a bacterium is infected by a virus, it uses a Cas nuclease to snip off a piece of viral DNA known as a protospacer. This fragment is stored in the bacterial genome with fragments from other viruses that have previously infected the cell - an immune memory. These viral spacer fragments are placed between repeated palindromic sequences, and this arrangement of spacers and palindromic repeats is what gives CRISPR its name.
Upon reinfection with the same virus, the bacterium can recognize and destroy it with Cas9. Cas9 activity relies on a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). The crRNA is complementary to the viral spacer that was stored after the original infection, while the tracrRNA serves as a scaffold; these two RNAs form a complex known as a guide RNA (gRNA). Think of the Cas9 as scissors, and the gRNA as the hand that’s directing them to cut.
Before cutting, the Cas9 acts as a search tool, checking the viral DNA for the protospacer adjacent motif (PAM), a short sequence downstream of the target site. When it recognizes PAM, Cas9 checks the region upstream - if it locates the target provided by the gRNA, it will create a double-stranded break (DSB). DSBs incapacitate the virus because viruses lack their own DNA repair mechanisms.
Once Dr. Doudna and Dr. Charpentier revealed the molecular mechanism that enabled the natural CRISPR-Cas9 system to cut DNA, the next question was obvious: if Cas9 is given a different guide RNA sequence, could it be used to create cuts at any desired location in an organism’s genome? Their theory was correct, and the results were groundbreaking.
The natural gRNA complex was engineered into a chimeric single guide RNA (sgRNA), offering a simple and cost-effective method of genetic manipulation. Researchers only needed to provide a different guide RNA - which can be generated with relative ease - and Cas9 could be used to create cuts at a range of target sites in the DNA of any organism (relying on the presence of the correct PAM sequence).
CRISPR-Cas9 gene editing works by creating double-stranded breaks in the DNA and then taking advantage of cellular DNA repair pathways. While there are several DNA repair pathways, the key ones used for gene editing are non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is exploited to render genes non-functional, while HDR is exploited to insert new genes or fragments of genetic material.
CRISPR is a powerful genome editing technology and has a wide range of applications. In this section, we’ll cover the various methods that make them possible. For more details on all of these techniques, you can check out our comprehensive, chapterwise look at methods here.
If Cas9 creates a DSB, it will most likely be repaired by NHEJ. However, NHEJ is error-prone, and it usually results in insertions and deletions (indels) in the region being repaired. When indels occur within the coding region of a gene and result in a frameshift mutation, the gene becomes non-functional. This is known as a gene knockout (KO).
Gene knockouts are used in a range of research areas, including functional genomics, pathway analysis, drug discovery and screening, and disease modeling. Using multiple guide RNAs that target various regions of the gene ensures high efficiency gene knockout, a method that is gaining popularity.
In the presence of a DSB induced by Cas9, cells can also repair themselves via HDR, and this pathway offers an opportunity for researchers to insert a new piece of DNA or an entire gene. This method is known as a gene knock-in.
To achieve a gene knock-in, there must be a DNA template for repair known as a donor template. This donor template consists of the sequence or gene of interest flanked by regions of homology that match the area on either side of the cut. The donor is delivered to the cells being engineered, along with the other editing components of Cas9 and sgRNA.
Gene knock-ins have been a key breakthrough in biotechnology, including the production of recombinant proteins, increasing the viability of immortalized cell lines, and precision disease modeling. Perhaps most importantly, CRISPR knock-in can be used in cell and gene therapies to correct genetic mutations that cause human disease.
Compared with gene knockout, gene knock-ins are more challenging. This is because HDR is a less common repair pathway than NHEJ, only occurring at certain stages of the cell cycle.The low frequency of HDR typically results in low knock-in efficiencies. However, scientists have been creating methods to overcome this obstacle, including detailed experimental optimization, cell cycle synchronization, and treatments that either boost HDR or disable NHEJ for knock-in experiments.
CRISPR-Cas9 can be used to delete (KO) or insert (KI) genes, but with slight modifications, it can also be used to regulate the expression of genes. This is known as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi). CRISPRa is used to increase (upregulate) the expression of a gene, while CRISPRi can reduce (downregulate) the expression of a gene.
Both of these technologies function by fusing an engineered variant of Cas9 known as catalytically dead Cas9 (dCas9) with transcriptional effectors to modulate target gene expression. dCas9 cannot cut the DNA, and so it simply leads the transcriptional effectors to the gene of interest.
These gene modulators have many research applications, including developmental biology, infectious disease, disease progression, functional genomics, and screening for genetic elements that mediate drug resistance.
CRISPR-Cas9 technology has enabled highly accurate, large-scale screening studies for drug discovery research, allowing us to more easily unravel the relationships between genotype and phenotype. CRISPR screens typically involve generating a large sgRNA library targeting different genes, making a wide range of edits to a particular cell line, then analyzing the effects of the edits on the phenotype of the cells.
By performing knockouts of multiple genes in a healthy cell line, researchers are able to identify which genes are involved in disease pathogenesis, while the same method in a diseased cell line can identify which genes are ideal targets for drugs. CRISPR screens provide more consistent results with less off-target effects than the RNAi screens that were previously employed for drug discovery. The multi-guide sgRNA knockout approach used by Synthego makes these screens even more reliable for target identification and validation.
Some of the most recently developed CRISPR methods are base editing and prime editing. These technologies work on the same principle, however at a more precise scale, inducing single nucleotide substitutions. Importantly, base editing and prime editing do not induce DSBs in the target DNA.
Unlike traditional CRISPR-Cas9 editing methods that introduce double-strand breaks, base editors enable targeted single-base conversions, reducing risks like insertions or deletions (indels) and chromosomal rearrangements. By chemically modifying DNA bases without cutting, base editing provides a safer and more efficient tool for addressing a wide range of genetic diseases. The base editor system consists of a deaminase enzyme fused to a modified Cas9 variant, such as a nickase (nCas9) where one catalytic site is active or a catalytically inactive Cas9 (dCas9), which is guided to the target site by a base editing gRNA. Once the deaminase removes the amino group (-NH2) from the target base, subsequent DNA replication or repair ensures the desired base change, making this approach highly specific and effective for correcting SNVs and generating stop codons that result in precise gene knockouts.
Two key types of base editors are cytosine base editors (CBEs) and adenine base editors (ABEs), each targeting different base pair conversions. These editors have demonstrated high efficiency in correcting disease-causing single nucleotide mutations (SNVs) such as those in genes linked to monogenic disorders. Despite their promise, challenges like off-target effects, varying efficiencies, and the need for advanced delivery systems remain areas of active research. With ongoing innovation, base editors are becoming indispensable tools in the development of cell and gene therapies, enabling precision medicine to address previously untreatable genetic diseases.
Learn more about Base Editing mechanisms and clinical trials.
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Prime editing offers unmatched precision for small insertions, deletions, and all 12 possible base conversions, surpassing the limitations of traditional CRISPR and base editing. Unlike earlier methods, it achieves these edits without introducing double-strand DNA breaks or requiring donor DNA templates, reducing risks like genomic instability. The system relies on a fusion of Cas9 nickase and reverse transcriptase, guided by a specialized prime editing guide RNA (pegRNA). The pegRNA directs the prime editor enabling it to make precise edits by synthesizing and incorporating new DNA sequences directly into the genome. This breakthrough addresses a wider range of genetic mutations with high specificity, paving the way for safer and more effective therapeutic applications.
Recent advancements in prime editing, such as PE3, PE3b, and EXPERT systems, have significantly improved editing efficiency and precision. These iterations introduced dual nicking strategies, refined pegRNA designs, and enhanced reverse transcriptase components, allowing for complex modifications such as large-fragment edits with minimal off-target effects. Prime editing faces challenges such as complex pegRNA chemical syntheses, delivery inefficiencies, and off-target effects, but innovative solutions are actively addressing these issues. Advances like optimized RNA chemical synthesis, advancements in delivery systems, and engineered high-fidelity Cas9 variants have significantly enhanced efficiency and precision. Continued innovations, such as mismatch repair inhibitors and scalable production methods, are paving the way for advancements in therapeutic applications using prime editors.
Learn more about Prime Editing mechanisms and clinical trials.
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In just a few short years, CRISPR has had a massive impact on scientific research, contributing to breakthroughs in medicine and biotechnology. Let’s take a closer look at some of the key applications of this technology.
CRISPR is poised to revolutionize medicine, with the potential to cure a range of genetic diseases, including neurodegenerative disease, blood disorders, cancer, and ocular disorders. As we mentioned earlier, the first trial of a CRISPR cell therapy was performed in 2019, treating patients with sickle cell disease. The treatment restored fetal hemoglobin, eliminating the need for a functional copy of adult hemoglobin.
In 2021, a significant CRISPR trial for transthyretin amyloidosis, a neurodegenerative disease, showed very promising results. It is also revolutionizing pediatric cancer treatment, described in this podcast interview with Shondra Miller from St Jude’s.
CRISPR can also be used to generate chimeric antigen receptor (CAR) T cells, a form of immunotherapy used to treat cancer. The T cells are extracted from patients and engineered to express chimeric antigen receptors before being re-injected into the body. The receptors allow the T cells to more efficiently target and destroy the specific type of cancer the patient suffers from.
While we are still in the early years of clinical trials, this technology could be used to treat thousands of genetic conditions in the future, including breast and ovarian cancer linked to BRCA mutations, Huntington’s disease, Tay-Sachs, beta-thalassemia, cystic fibrosis, and early-onset Alzheimer’s. For all the latest medical developments and clinical trials using this technology to cure a range of human diseases, you can take a look at the CRISPR Medicine News website.
During the COVID 19 pandemic, CRISPR was used as both a potential therapeutics tool and as a diagnostic tool for the coronavirus. The SHERLOCK™ CRISPR SARS-CoV-2 test kit was granted Emergency Use Authorization from the federal authorities to be used in laboratory settings. You can learn more about SHERLOCK and the more recently developed STOPCovid diagnostic test here and in this podcast.
Mammoth Biosciences has also developed a CRISPR-based Covid-19 diagnostic method, known as DETECTR. Like SHERLOCK and STOPCovid, DETECTR utilizes Cas9’s search function to detect genetic material from the virus, employing naturally occurring Cas nucleases, like Cas12 and Cas13. For more information on DETECTR, you can listen to this interview with Trevor Martin, CEO of Mammoth Bioscience.
Similar diagnostics utilizing the search function of Cas9 have also been engineered to identify other diseases, both infectious and genetic. Early in 2021, Dr. Kiana Aran of Cardea Bio published a study which combined three different Nobel Prize-winning technologies - graphene, transistors, and CRISPR - into a tiny chip that can detect pathogenic single nucleotide polymorphisms (SNPs). Since 50% of disease-causing mutations in humans are SNPs, this is a significant breakthrough in medical diagnostics.
Gene editing technology has huge potential in agriculture, and experts suggest that CRISPR-modified foods will be available within 5-10 years. This is primarily because it can be used to create crops that are disease-resistant and drought-resistant. For example, scientists from the University of Berkeley and Innovative Genomics Institute have partnered with Mars, Inc. to create disease-resistant cacao plants.
It can also be used to prolong the shelf-life of other perishable foods, reducing food waste and allowing access to healthy foods at relatively low cost. For more information on these applications, you can read our overview of CRISPR’s use in agriculture.
As one of the leading alternatives to fossil fuels, bioenergy has been under the spotlight for a while now. However, there are several hurdles to producing biofuels at scale. By using CRISPR, scientists have recently been able to make some significant advances in this area.
For example, KO of multiple transcription factors that control production of lipids in algae has led to a huge increase in lipid production for generating biodiesel. Similarly, gene editing can improve the tolerance of yeast to harsh conditions during the production of biofuels. It has also increased editing efficiencies in bacterial species that are used to produce ethanol. For more details, you can check out this blog on how CRISPR is helping the biofuel industry.
The CRISPR system has enabled a faster and cheaper production of animal models compared with traditional gene-targeting methods, and can provide much more precise models of disease. Let's take a look at gene editing progress with respect to cell and animal models.
CRISPR editing has been a game-changer for stem cell research. Stem cells are pluripotent, meaning that they can differentiate into any type of cell. This makes them incredibly valuable in medical research, however harvesting stem cells from embryos is highly controversial. The advent of induced pluripotent stem cells (iPSCs) was a major breakthrough, because they are adult cells (like skin or blood cells) that have been reprogrammed to become pluripotent.
While human iPSCs avoid the ethical concerns of embryonic stem cells, they remain challenging to work with - not only is it difficult to reprogram and grow iPSCs, but they are also quite resistant to genetic manipulation. CRISPR has not only made reprogramming iPSCs easier, but has also delivered much more efficient results than previous gene editing technologies which were originally used to engineer iPSCs.
CRISPR-edited iPSCs have been successfully developed as cell therapies, like the CTX001 trial for sickle cell disease we mentioned earlier, and large-scale disease modeling studies, such as the 2021 Inducible Pluripotent Stem Cell Neurodegeneration Initiative (iNDI). Recently, CRISPR was used to generate triple-knockout, 'off-the-shelf' CAR T cells which are hypoimmunogenic, meaning that they can be used to treat any patient - a massive breakthrough for T cell immunotherapy.
CRISPR-Cas9 edited iPSCs can also be used to generate organoids - tiny, three-dimensional models of human organs that can be grown in the lab. Organoids are more relevant to the study of human disease than cell cultures that are maintained as monolayers or suspensions, because they provide more of the complexity of real human organs and can therefore more accurately mimic disease progression and response to treatment. Recently, edited iPSCs were used to generate brain organoids to study how the human brain evolved.
Primary human cells are incredibly valuable for disease modeling as they provide more accurate biological data than immortalized cell lines, and gene-edited primary cells can be used for both disease modeling and cell therapy. However, successful culturing and genetic manipulation of primary cells have been significant challenges in biomedical research.
In recent years, scientists have provided solutions to increase CRISPR editing efficiency in primary cells, including synchronizing the cell cycle, the use of chemically modified sgRNAs, creating ribonucleoprotein (RNP) complexes for delivery, and optimizing delivery methods based on the specific cell type. These advances have led to many successful cell therapies, including T cell immunotherapies.
Traditional animal models of human disease are useful, but they have several limitations, including a lack of translational capacity. With CRISPR, however, creating transgenic animals that more accurately recapitulate human disease states has become significantly easier. CRISPR editing in animals is much more efficient than previous genome engineering technologies - it can generate almost any required edit, as well as having the capacity to generate multiple edits within the same organism.
CRISPR can be used to generate 'humanization knock-ins' in animals like mice - deleting a particular gene or region of DNA in the animal and replacing it with the human version. This level of accuracy is key to understanding and treating human disease. For example, gene-edited animal models of Duchenne Muscular Dystrophy have recently led to significant breakthroughs in treating this disease, including the development of a potential gene therapy, experimentally demonstrated in dogs. It has also been used to create large animal models of neurodegenerative disorders which may lead to clinical trials in the near future.
Now that you're more familiar with the basics of CRISPR and its applications, you're probably curious about how to use it in your own research. In this section, we'll take you through the main steps of designing your own experiment. A more detailed chapter-by-chapter guide for designing your gene editing experiment is also available.
Guide RNA design is one of the most crucial aspects of designing a successful CRISPR experiment. The choice of sgRNA will very much depend on the type of edit you require, whether it be KO, KI, base or prime editing, CRISPRi, or CRISPRa.
Synthego's CRISPR Design Tool is a free resource available to all researchers globally, enabling the design and selection of guide RNA sequences to target any gene across over 120,000 genomes. Synthego also has an in-depth guide to choosing the right gRNA for your experiment.
The choice of Cas nuclease is also important for the success of your experiment. Cas9 was the first and most commonly used Cas nuclease, originally isolated from the bacterial strain Streptococcus pyogenes (SpCas9). However, there are now many different naturally occurring and engineered Cas variants available for different purposes.
For example, engineered Cas9 nickases are used to create single-stranded breaks and high-fidelity Cas9 variants have been engineered to reduce off-target edits, while naturally-occurring Cas 13 is used for its diagnostic potential.
There is no universal transfection protocol for all experiments, because delivery of editing components depends on the cell and organism you're targeting, as well as the type of editing cargo being delivered. There are various transfection protocols available, and you can also check out our in-depth blog on delivery methods.
Researchers at Synthego have determined that the best method for introducing gene editing components into the majority of cell types involves the nucleofection of ribonucleoproteins (RNP) complexes consisting of chemically modified synthetic sgRNA and purified recombinant Cas9.
The last step in evaluating your experiment is picking an analytics tool to confirm that editing was successful. Like every other step, this must be tailored to the experiment. The gold standard is to use Next Generation sequencing (NGS), however this is not always in every lab's budget, especially if you need to sequence frequently or have many samples to run.
Synthego's Inference of CRISPR Edits (ICE) is an online tool for analyzing Sanger sequencing data, which is cheaper than NGS. ICE is user friendly, open-source, and highly accurate, with results comparable to NGS.
There can be no doubt that CRISPR-Cas9 has revolutionized the field of genome engineering. However, we’re only just beginning to see the benefits and possibilities of this incredible technology - with a variety of successful preclinical studies and more clinical trials being approved, the dream of curing human disease by editing our DNA is now very real.
There are also an increasing number of biotech startup companies focusing on CRISPR-Cas9 gene editing technology, and many researchers are continually finding new ways of applying this technology to solve real-world problems, including epigenome editing, new cell and gene therapies, infectious disease research, and for the conservation of endangered species.
CRISPR has been widely adopted by researchers around the world, resulting in thousands of publications. In this section, we’ll explore some exciting papers that utilize this technology, including a few significant breakthroughs.
1. Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells
This recent study performed Cas13-based RNA editing on human cells, partnering with Synthego to experiment with different types of chemical modifications to the guide RNAs to determine which could provide superior knockdown efficiencies.
2. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
In an effort to reduce off-target edits, this study engineered a high-fidelity Cas9 variant, SpCas9-HF1. The novel nuclease retained all the on-target efficiency of wild-type Cas9 with no detectable off-target edits.
3. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
David R Liu and his team at Harvard demonstrated the first use of CRISPR as a base editor, substituting one base for another using a Cas9 nickase and without creating DSBs.
This 2015 paper demonstrates the use of CRISPR as an epigenome editing tool. By fusing dCas9 to the human acetyltransferase p300, the authors generated highly specific edits, activating genes of interest.
5. Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms
This 2020 paper published in Science performed knockout screens in the human Caco-2 cell line, examining the effects of KO on the ability of SARS-CoV-2 to infect human cells. Using this method, the authors were able to identify potential targets for treatment of Covid-19.
Published in Nature Medicine, this study was a breakthrough in T cell immunotherapy. By disrupting the programmed cell death 1 (PD-1) gene, Tony Mok and his team were able to improve the efficacy of T cell therapy in patients with non-small-cell lung cancer (NSCLC).
7. In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials
By engineering bacteriophage (crPhage) to express Cas3, the authors of this study were able to target and kill pathogenic bacteria by deleting large regions of their DNA. This strategy is suggested as an alternative approach to combat the rise of antibiotic-resistant bacteria.
We hope this guide has helped you gain a better understanding of CRISPR-Cas9 gene editing, how it can be used, and the wider implications of this technology. With its rapid adoption in laboratories across the globe and an ever-increasing range of applications, CRISPR has certainly revolutionized science, from basic research to medical biotechnology. For a more in-depth guide, you can check out our downloadable CRISPR 101 ebook, or to start using this technology in your research, you can book an appointment with one of our genome engineering specialists.
CRISPR has quickly become a standard laboratory tool for gene editing. As the adoption of CRISPR accelerates worldwide, up-to-date knowledge of the basics of CRISPR is essential for anyone in the field. From target identification studies to the recent breakthroughs in clinical trials, CRISPR is enabling scientists to unlock the power of the genome.
Download our CRISPR 101 eBook today to stay up to date on all your CRISPR basics and get the best results in your CRISPR experiments!
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