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As a global leader in gene synthesis and genome editing, GenScript offers reliable and efficient genome engineering tools to researchers worldwide. Through our strategic partnership with Feng Zhang's laboratory at the Broad Institute, GenScript CRISPR technology simplifies gene editing with reliable reagents, user-friendly online design tools, optimized protocols, and expert technical support. With GenScript's CRISPR services, you can seamlessly harness the full power of genome editing for groundbreaking research and discoveries.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences were originally identified in the Escherichia coli (E. coli) genome, and were found to function as part of an RNA-based adaptive immune system to target and destroy genetic parasites at the DNA level.
CRISPR systems have since been adapted for use in various eukaryotic cell types as a simple but versatile genome editing tool, allowing researchers to make specific changes to a cell’s DNA.
The most basic CRISPR gene editing experiment, gene knockout, requires two components to be delivered to the cell’s nucleus; a targeted guide RNA and a Cas nuclease. These components form a complex that binds DNA at the target sequence and generates a double-strand break (DSB), creating an opportunity for knockout.
Guide RNA
CRISPR guide RNAs are designed to target specific DNA sequences near a protospacer adjacent motif (PAM) site and may be used as single guide RNA (sgRNA) or as a duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
GenCRISPR guide RNAs are available as synthetic sgRNA or plasmid crRNA.
Cas Nuclease
CRISPR associated protein (Cas) are RNA-guided nucleases capable of cleaving the phosphodiester bonds of a DNA backbone to create a double-strand break. The most commonly used Cas for CRISPR engineering is Cas9 from S. pyogenes (SpCas9). Cas9 orthologs have also been discovered from S. aureus (SaCas9) and C. jejuni (CjCas9). Additional naturally occurring Cas nucleases include Cas12a, Cas12c, Cas12e, Cas13a, and CasX. Variations in PAM specificity among these Cas variants will affect guide design flexibility and the potential for incidental off-target editing.
In addition to the wild-type Cas nucleases, researchers have engineered variants for specific uses, including improved editing specificity (eSpCas9) and relaxed PAM specificity (SpCas9-NG). Researchers have also altered Cas9 nuclease activity for single-strand cutting (nCas9) or deactivated it entirely (dCas9) for use with transcriptional activators or repressors. For more information on these alternative CRISPR-Cas systems, please see the CRISPR Toolbox section below.
GenCRISPR Cas Nucleases are available as protein or mRNA.
Delivery
Both CRISPR guide RNA and Cas nuclease must be delivered into the cell nucleus for successful editing to occur. CRISPR/Cas delivery strategies may be separated into two types; non-viral transfection and viral vector transduction. The chosen strategy will largely determine which reagent formats are suitable. For more information, see the Engineering Workflows section.
| Non-viral transfection | Viral vector transduction |
|---|---|
| Deliver CRISPR components as RNA or protein directly into cell nucleus | Deliver virus packaged plasmids encoding CRISPR components for integration |
| Electroporation, lipofection, microinjection, biolistics | Lentivirus, AAV |
Multiple delivery strategies may also be used in series, for example by first transducing lentiviral packaged Cas9 plasmid to generate a stable Cas9 expressing cell line, and then electroporating guide RNAs for editing.
Editing & Repair
Once Cas9 nucleases are guided to the target DNA and create a DSB 3-4 bases upstream from the PAM sequences, there are two ways the DSB can be repaired.
If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel formation that effectively knocks out protein function in some percentage of the cell population.
Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.
The most common CRISPR engineering workflows include:
| Workflow | sgRNA | Cas | Recommended Delivery | Description | Ideal for | Benefits |
|---|---|---|---|---|---|---|
| RNP | synthetic | protein | Transfection (electroporation, lipofection) | Synthetic CRISPR sgRNA and Cas protein are complexed as ribonucleoprotein (RNP) prior to delivery. | ex vivo editing, embryonic microinjection | DNA-free / transgene-free. Editing begins immediately. |
| mRNA | synthetic | mRNA | Co-transfection | Synthetic CRISPR sgRNA and Cas mRNA are co-delivered. | in vivo editing | DNA-free / transgene-free. Editing begins post-Cas9 expression. |
| Co-plasmid | plasmid | plasmid | Viral packaging | Separate viral packaged plasmids containing CRISPR sgRNA and Cas are co-delivered, and randomly integrate for constitutive expression. | Difficult-to-transfect cell lines | Simple delivery and selection/enrichment. |
| All-in-one | single plasmid | single plasmid | Viral packaging | Single viral packaged plasmid containing CRISPR sgRNA and Cas is delivered, and randomly integrates for constitutive expression. | Difficult-to-transfect cell lines | Single viral transduction delivery step |
| Stable Cas9 cell line | synthetic | plasmid | Viral Cas plasmid, transfected sgRNA | Viral packaged Cas plasmid is delivered to generate stable Cas-expressing cell line. Then synthetic sgRNA is delivered for editing. | Screening | Easily test stable Cas9 expressing line with various gRNA |
Traditional Gene Knockout
As described above, the CRISPR-Cas complex generates a targeted double-strand break, creating an opportunity for gene editing.
Gene knockout (KO) experiments exploit flaws in the cell’s native repair mechanism, non-homologous end joining (NHEJ). Imperfectly repaired genes with insertions or deletions of base pairs will result in frameshift mutations, rendering the gene and its corresponding protein non-functional. KO guides are algorithmically designed and ranked for both specificity (low homology with any other genomic sequence) and functionality (targeting a portion of the gene most likely to result in functional protein KO). CRISPR KO can be applied in many research scenarios, from basic screening, functional studies of a gene, bioengineering, agricultural biotechnology, to developing therapeutic gene and cell therapeutic drugs.
Increasing CRISPR/Cas9 Editing Specificity
CRISPR Cas9 based gene knockout relies on the specificity of both the sgRNA and the Cas9 protein. Poorly designed sgRNA and traditional wildtype Cas9 would lead to editing at undesired genome locations, which are considered as off-target edits. Many Cas9 variants have been developed for enhanced specificity. For example, the eSpCas9(1.1), also referred to as SpCas9 (K848A/K1003A/R1060A), structurally engineered from Feng Zhang lab contains alanine mutations that weaken the bounding between the HNH/RuvC groove and the none targeting DNA strand, reducing off-target effects by over 10-fold while maintaining robust on-target genome editing efficiency (Slaymaker et al. 2016).
Researchers have also engineered CRISPR Cas variants, such as Cas9 nickases, to “nick” a single DNA strand instead of creating double-stranded DNA breaks, enhancing editing precision. Two individual nickases targeting opposite DNA strands are necessary to generate a break site for gene editing, which significantly increases targeting specificity. For example, SpCas9 nickase (Cas9n D10A) contains a mutation allowing the endonuclease to create single-strand nicks, as opposed to DSBs. Pairing two opposite facing gRNA sequences with SpCas9 nickase can be an efficient method of gene editing that prevents unwanted indels from forming.
Novel Editing Technologies Without DSBs (Prime Editing/Base Editing)
Cas9 nickases also could enable novel single-strand CRISPR gene editing techniques that avoid the potential for unintended genomic changes posed by double-strand DNA breaks. Prime editing utilizes a Cas nickase protein fused to a reverse transcriptase (RT) to write a new sequence into a target DNA site directly. Prime editing guide RNAs (pegRNA) contain both a DNA targeting sequence and an RT template so that the pegRNA-dCas9 nickase complex can identify the target site, cut a single strand, and write the new sequence. Multiple versions of the prime editing system have been developed to improve editing efficiency.
Base editing utilizes a Cas9 nickase, or alternatively a catalytically dead Cas9 (dCas9) which only binds to target DNA without cutting, fused to a nucleobase deaminase enzyme and a DNA glycosylase inhibitor to make targeted point mutations. For example, cytosine base editors (CBEs) and adenine base editors (ABEs), by fusing Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC or adenine DNA deaminases, can convert C to T (or G to A) and A to G (or T to C).
Traditional Gene Knockin
Gene knock-ins have various applications in biotechnology, including disease modeling and gene and cell therapy applications. CRISPR based gene insertion relies on a pathway different than NHEJ, it is called Homology directed repair (HDR) pathway. HDR process is very precise allowing for accurate gene knockin, but HDR occurs in less frequency compared to NHEJ leading to less editing efficiency compared to knockout. In these experiments, the Cas9 cut site plays a critical role, as it determines where the repair process occurs. Successful HDR also requires the introduction of Donor DNA HDR templates, which can be single- or double-stranded DNA designed with overlapping homology arms to the specific Cas9 cut site for precise gene integration.
To increase the efficiency of HDR, experiment optimization by testing different template format, delivery approaches, culture conditions, as well as using small molecules for cell cycle synchronization, enhance HDR or inhibit NHEJ have been used to overcome this challenge.
Read more on HDR template formats available and design.
CRISPR Transcriptional Regulation
Additional Cas9 variants have been developed for transcriptional regulation of gene expression. Rather than editing DNA, these deactivated Cas9 proteins simply bind the target site and activate or inhibit expression.
CRISPR activation (CRISPRa) systems utilize a dCas9 fused to transcriptional activators to up regulate endogenous gene expression, whereas CRISPR interference (CRISPRi) systems use dCas9 fused to transcriptional repressors to downregulate gene expression. Multiple CRISPRa and CRISPRi systems have been developed using different activators or repressors. CRISPRa gRNAs for the SAM system are currently available in plasmid formats.
Researchers have also recently developed a Cas fusion protein, CRISPRoff, that can epigentically modify and silence genes without editing the underlying genome. The epigenetic memory persists through cell differentiation and is heritably passed to future cell generations. Gene activity can be re-activated using a different fusion protein called CRISPRon.
| Technique | Components Required | Common Experiments | Common Applications |
|---|---|---|---|
| CRISPRa | CRISPRa guide RNA + dCas9-SAM (or other) |
Overexpression screening and hit analysis | Drug discovery |
| CRISPRi | CRISPRi guide RNA + dCas9-KRAB (or other) |
Transient loss-of-function, siRNA validation | Drug discovery |
| CRISPRoff/ CRISPRon |
CRISPRoff guide RNA + CRISPRoff | Heritable gene silencing without DNA editing | Gene therapy |
Though Cas9 protein has been widely popular and used in various research projects, did you know that there are many other types of Cas proteins that might be even better suited for your specific research needs?
CRISPR/Cas systems can be categorized into two classes, Class I and Class II, based on differences in the organizations of effector modules. Research suggests that all CRISPR/Cas systems may have originated from a common ancestor. In this evolutionary process, class I systems were encoded by a single-function cas gene and lost some additional cas genes over time, forming class II systems. These classes are further divided into six types: type I to type VI. Class I includes type I, III, and IV, while class II includes type II, V, and VI (Table 1). Different types of CRISPR/Cas systems have different functions based on the structure and function of their Cas proteins. Type I, II, and V systems are involved in recognizing and cleaving DNA, while type VI can edit RNA. Type III has the ability to edit both DNA and RNA. As for the effect of type IV systems on DNA or RNA, it is still not fully understood and requires further study. By understanding the different classes and types of CRISPR/Cas systems, we can better appreciate the diversity and potential applications of this technology. Here we will highlight Cas12 and Cas13 as the two other commonly used Cas protein types used in research now than Cas9.
| Class | Type | Effector/Protein | Target Molecule | Need tracrRNA | PAM/PFS |
|---|---|---|---|---|---|
| 1 | I | Cascade (e.g. Cas3) | DNA | No | - |
| III | Cascade (e.g.Cas10) | DNA/RNA? | No | - | |
| IV | Cascade (Csf1) | DNA | No | - | |
| 2 | II | Cas9 | DNA | Yes | SpCas9 & FnCas9 uses ′NGG’ |
| V | Cas12 | DNA | Yes/No | TTN or TTTN | |
| VI | Cas13 | RNA | No | A, U, or C |
Table 1: Summarization of commonly used Cas protein types and characteristics.
Cas12
There are 3 subtypes under Cas12, including Cas12a, Cas12b, and Cas12c, among which Cas12a is the most popular. Cas12a, as known as Cpf1, distinguishes itself from Cas9 through several key characteristics:
Cas13
Unless Cas9 and Cas12, Cas13 family is specific for targeting RNA sequences. Currently, there have been four subtypes of Cas13 that have been characterized: Cas13a, Cas13b, Cas13c, and Cas13d. Cas13 protein sizes range from ~900-1250 amino acids, and Cas13 guide RNA consists of a conserved hairpin-like structure of around 70 nucleotides and a variable region of 29 nucleotides that is complementary to the target RNA. Once activated, Cas13 exhibits nonspecific RNase acidity and cleaves all nearby RNA molecules regardless of sequence, which is leveraged in diagnostic tool development, for example Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). In addition, Cas13 is also used widely for RNA knockdown in eukaryotic cells, as well as RNA tracking/imaging and modification via using an catalytically inactive Cas13 (dCas13) which only bind, but not cleave RNA.
Guide RNAs are the foundation of any CRISPR-based gene editing experiment, therefore the design of gRNAs is a critical step in ensuring successful outcomes. To maximize the efficiency of edits and avoid off-target effects, careful considerations should be given to the following key areas in gRNA design:
sgRNA for CRISPR KI can be designed using different tools available online, for example GenScript FREE Online HDR Design Tool. The principles of choosing appropriate sgRNA sequences are:
HDR template can also be designed with our newly launched FREE Online HDR Design Tool
2.1 Key considerations for selecting template format
2.2 Key considerations for designing an HDR template
2.3 Additional benefits and strategies for Cas9 targeting sequence (CTS) design
Dr. Marson's group at the Gladstone-UCSF Institute of Genomic Immunology designed a hybrid HDR template structure with Cas9 target sequences (CTSs), allowing for the formation of a complex with RNP that can be delivered more easily to the nucleus. CTS design can be added to linear HDR DNA templates like GenExact ssDNA, and have been shown to improve HDR efficiency to >80-90% across a variety of target loci and primary human cell types. To learn more, download Design and Preparation Guidelines for CRISPR KI HDR Templates.
Achieving successful gene editing requires the efficient delivery of CRISPR components into cells. Various methods exist for delivering CRISPR into cells, including viral-based approaches and non-viral strategies. In this discussion, we will primarily focus on commonly used non-viral methods for delivering CRISPR ribonucleoprotein (RNP) into mammalian cells, considering the growing popularity of CRISPR RNP in both preclinical and clinical studies.
Electroporation, which uses short pulses of electrical current to temporarily increase cell permeability. This allows molecules, including the CRISPR RNP as well as other DNA/RNA or else, to enter the cell ex vivo. Electroporation has been shown to enhance gene editing efficiency and is now commonly used for editing genomes in primary cells or cells that are difficult to transfect. Commonly used electroporation systems include Nucleofector® System (Lonza), Neon™ Transfection System (Thermo Fisher Scientific), and Flow Electroporation® (MaxCyte).
Lipofection is another delivery strategy where lipid molecules surround the molecule to be delivered, forming a complex called a liposome. The liposome can fuse with the cell's plasma membrane and release its contents into the cell. Lipofection protocols that are already known to have high efficiency in delivering non-RNP molecules, such as plasmids, and also work for RNP delivery. Though it is delivery efficiency in most cases are not as high as electroporation.
Microinjection is a labor-intensive and time-consuming technique used for oocytes and embryos. It involves injecting the CRISPR components directly into individual embryos which requires skilled lab workers and specialized equipment, restricting its common usage to animal disease model generation projects.
Nanoparticles, small particles made of various materials, hold promise for delivering CRISPR components in vivo. Gold nanoparticles, for example, have been successfully used to deliver RNPs to the brains of mice. Other materials, such as zinc ions, are also being explored for nanoparticle-based delivery.
New approaches editing ex vivo are also being investigated. For example, GenScript is partnering with Indee labs on optimizing a novel non-viral method Hydropore™, which utilizes the power of microfluidic vortex shedding to deliver molecules including both nucleic acids and proteins to immune cells. Similarly, we are also collaborating with Avectas on testing their Solupore system which delivers cargo by physicochemical means for cell editing.
Additionally, we are partnering with Avectas to evaluate Solupore™, an advanced non-viral gene delivery platform that utilizes physicochemical mechanisms to efficiently transport nucleic acids and proteins into cells. This innovative technology enhances precision genome editing, improves cell viability, and optimizes ex vivo cell engineering workflows, making it ideal for applications in cell therapy and immunotherapy development.
CRISPR gene editing is powering a new generation of immuno-oncology cell therapies, allowing researchers to engineer immune cells with specific cancer-fighting properties. Specifically, CRISPR systems create targeted breaks in the immune cell genome, where engineered DNA templates can be inserted via the cell’s innate repair pathways. Armed with these new genetic inserts, immune cells can identify and target cancer cells with greatly improved effects.
While traditional immune cell therapies have relied on viral vector systems to deliver CRISPR components and DNA templates, safety concerns and manufacturing bottlenecks have shifted focus to non-viral approaches. 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. Ex vivo non-viral CRISPR ribonucleoprotein (RNP) systems offer a safe, efficient, and inexpensive immune cell therapy alternative. CRISPR guide RNA and Cas nuclease protein templates are complexed as RNPs before being co-delivered with DNA repair templates via electroporation or lipofection into T or NK immune cells, which are then reinjected into the patient.
Here we examine how researchers apply non-viral CRISPR RNP gene editing to develop novel immune cell therapies.
T Cells
CRISPR RNP gene knock-in techniques are currently being used to construct transgenic chimeric antigen receptor T (CAR-T) cells and T cell receptor T (TCR-T) cells for immunotherapy. Yet challenges remain to improve editing efficiency while reducing cytotoxicity.
Recently, significant efficiency improvements have been achieved using non-toxic GenExact single-stranded DNA (ssDNA) templates designed with a Cas9 Targeting Sequence (CTS) to bind the RNP for co-delivery via electroporation1. This method achieved >90% knock-in efficiency with minimal cytotoxicity in multiple primary immune cell types.
NK Cells
NK cells are another attractive target for immune cell therapy. However, their highly sensitive and resistance to exogenous DNA limits the effectiveness of viral and plasmid-based approaches.
Non-viral CRISPR RNP systems are currently being optimized to engineer chimeric antigen receptor NK (CAR-NK) cells to improve cancer-killing activity and reduce tumor evasion2-6. Non-viral approaches using Cas9 mRNA have also been applied to NK cell engineering to achieve >98% transfection efficiency with >90% cell viability7.
B Cells
Antibody-producing B cells are another exciting target for immune cell therapy. While initial research focused on AAV-delivered antibody-expressing cassettes, B cells often produced insufficient antibody expression levels or duration8.
Therefore, CRISPR RNP-based workflows are currently being optimized for applications in B cell therapy9,10. Specifically, targeted DNA insertion at the B cell receptor loci allows researchers to reprogram antibody specificity11.
CRISPR gene editing therapies treat genetic disorders at their source, by knocking out harmful genes or knocking in repair sequences, offering the potential to cure a wide variety of genetic disorders, including primary immunodeficiencies, leukodystrophies, hemoglobinopathies, and lysosomal storage disorders.
Traditional gene therapies have relied on viral vectors to deliver therapeutic transgenes. However, viral systems carry safety risks of insertional oncogenesis and immunogenic toxicity1, as well as high manufacturing costs. 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.
Here we examine some of the ways researchers are applying non-viral CRISPR RNP gene editing in HSPCs to develop ex vivo therapies for genetic disorders.
Primary Immunodeficiencies
Primary immune deficiency diseases (PIDDs) comprise a large and diverse group of 450+ chronic immune system disorders caused by hereditary genetic defects. PIDDs differ significantly by onset, symptoms, severity, and organs or tissues affected, but all result from impaired immune system function due to an underlying mutation.
While PIDDs have traditionally been treated via cell and gene replacement methods, such as allogeneic stem cell transplantation or viral-based autologous gene therapies, CRISPR RNP genome editing therapies can efficiently correct the defective gene without concerns for donor-matching or viral transgene integration 2,3.
For example, CRISPR RNP editing in HSPCs has demonstrated therapeutic potential for the correction of SCID-X14, XHIM 5, X-GCD 6, and WAS 7. Researchers have also achieved high-efficiency gene insertion of >80-90% using ssDNA repair templates to correct IL2RA and CTLA4 mutations associated with immunodeficiencies 8.
Hemoglobinopathies
Hemoglobinopathies are hereditary disorders affecting red blood cell structure (sickle cell diseases) or production (thalassemias). These disorders are caused by single-gene mutations, making them ideal candidates for CRISPR gene editing therapies 9,10.
Non-viral CRISPR RNP editing has been used to restore β-globin expression in HSPCs from β-thalassemia patients 11, and to knock-in single-stranded DNA (ssDNA) repair templates in HSPCs from sickle cell patients 12. Companies such as CRISPR Therapeutics are currently developing CRISPR RNP gene editing therapies for both types of hemoglobinopathies.
Lysosomal Storage Disorders
Lysosomal storage disorders (LSDs) comprise a group of 70+ rare progressive metabolic diseases, in which mutations to lysosome related enzymes interfere with proper cellular metabolism and cause toxic buildup of fats or sugars in the cell. Targeting these mutations with ex vivo CRISPR gene editing therapies offers great promise for potential treatments 13, 14, 15.
Specifically, CRISPR RNP editing has been used to insert DNA templates to correct the mutations associated with Batten Disease in patient-derived iPSCs 16 and Mucopolysaccharidosis type I 17 and Gaucher Disease 18 in HSPCs. Additionally, RNP editing in HSPCs has demonstrated therapeutic potential in metachromatic leukodystrophy patients 19.
CRISPR screening is a powerful technique used to identify important genes within a vast number of genetic sequences, such as the entire genome. It can pinpoint genes that influence various physiological effects, including drug resistance and sensitivity. CRISPR-Cas9 is particularly useful for target-specific gene modifications, allowing researchers to identify drug targets or validate and optimize existing ones. By combining CRISPR with next generation sequencing and single-cell sequencing technologies, scientists can perform whole-genome analysis and make significant advancements in identifying disease-specific targets.
Figure 1: How to use pooled CRISPR gRNA libraries
The basic principle behind CRISPR screening involves knocking out individual genes in a mixed population of cells to determine their impact on cell survival and growth. CRISPR screening begins with the knockout of one gene per cell via using a CRISPR library contains a pool of lentivirus each possess gene encoding a different gRNA, resulting in a population of cells with different genes knocked out. These cells are then pooled together and allowed to grow for a few days for NGS to identify which genetic sequences are present or knocked out. CRISPR screening libraries are commercially available and enable scientists to analyze specific gene families or pathways by creating targeted gene knockouts and studying the resulting phenotypic alterations.
In certain cases, researchers may have a short list of genes of interest and use an arrayed gRNA CRISPR library. This library consists of synthetic sgRNA kept in separate wells on a multi-well plate. Each well contains one or several predetermined CRISPR targeting sequences that are transfected into cells. This approach eliminates the need for next-generation sequencing but requires automation equipment and is more suitable for screens focused on phenotype changes caused by gene knockout.
Scientific research constantly seeks to unravel the complexities of various diseases and develop effective treatments. One crucial aspect of this pursuit is the generation of disease models, which mimic specific disease to serve as invaluable tools for researchers to study disease mechanisms, gain insights into the underlying biology, and aiding drug discovery and development. There are different types of disease models, each with its advantages and limitations:
In vitro disease models, such as cell cultures or organoids, involve growing cells in a laboratory dish or three-dimensional culture system and allow studying specific cellular processes and test the effects of drugs or genetic modifications.
Patient-derived disease models, such as induced pluripotent stem cells (iPSCs) generated from patient samples. These models allow for personalized medicine approaches, as they reflect the genetic and cellular characteristics of an individual patient's disease.
Animal models, including rodents, primates, and other organisms, are widely used to simulate complex diseases. These models often involve genetically modified animals or the transplantation of human cells or tissues into animals, allowing investigation of disease progression, interactions between different organ systems, and the potential systemic effects of interventions.
CRISPR technology has been adopted for developing engineered immortalized cell lines, iPSCs, as well as animal models in the past 10 years, and made tremendous progress compared to traditional approaches. For cell line engineering, gRNA and Cas9 (and donor template if doing knock-in or mutation) will be delivered into cell lines using lentivirus or electroporation, whereas for animal models, the genome editing components are commonly microinjected into fertilized mouse eggs, circumventing the need for enrichment and selection offered by mouse ESCs.
Choosing the right CRISPR reagents for the generation of CRISPR-edited mice. Payload size is the key driver for determining which donor template format is best. ssDNA works best for shorter payloads (<5kb), while plasmids are suitable for the larger payloads. CRISPR machinery (guide and Cas endonuclease) can either be delivered as a fully formed RNP complex, or as a separate guide and an encoding Cas messenger RNA (mRNA). In general, RNP offers more consistency in generating non-mosaic animals and a lower risk of off-target effects. After CRISPR-edited mice have been generated, off-target effects can be screened using Sanger sequencing of individual predicted sites, or using NGS-based methods for genome-wide analysis
CRISPR is easier and much cheaper than conventional methods especially for animal model generation. For example, CRISPR approach only need roughly 3 months and $5,000 to generate founder mice, compared to 8-10 months and up to $50,000 using embryonic stem cell (ESC). CRISPR also achieves higher success rate compared to ESC for generating KO animal model, 85% vs 50%; as for KI, the success rate may vary depend on the mutation or insertion size and gene characteristics, and donor template format. In comparison to dsDNA and plasmid DNA, ssDNA has shown the best editing efficiency in mouse model generation. Read more about using ssDNA for transgenic mice development.
CRISPR is a powerful tool for precisely editing DNA sequences in living cells, allowing researchers to study gene function and develop novel therapies for cancer and genetic diseases. While CRISPR is most commonly associated with gene editing, it also has a range of other potential applications, including in diagnostic testing.
Diagnostic testing is a critical component of modern medicine, allowing doctors to identify and treat diseases in their early stages. Traditional diagnostic methods, such as PCR and ELISA, can be time-consuming, error-prone, and often require specialized equipment. However, CRISPR-based diagnostic techniques can offer a more efficient and accurate approach to disease detection in the field or at point-of-care. CRISPR-based nucleic acid detection assays detect specific RNA or DNA sequences associated with a particular disease or pathogen, with potential applications in infectious disease diagnosis, genetic testing, and cancer detection.
How CRISPR Diagnostics Work
The principle behind these assays is simple: if the target sequence is present in a sample, the CRISPR system cleaves a reporter molecule, producing a signal to indicate the presence of the target sequence. Standard signal methods such as colorimetric, fluorescence, and lateral flow detection can be used in CRISPR-based diagnostic systems. The main advantages of CRISPR-based nucleic acid detection assays are their sensitivity and flexibility. These assays can detect minimal amounts of genetic material, making them ideal for detecting diseases in their early stages.
Additionally, some CRISPR-based diagnostic systems require minimal sample preparation, reducing the cost and time needed for diagnostic testing. This allows for testing in the field with a portable device, making CRISPR assays ideal for use in remote and resource-limited settings. Finally, because CRISPR can be programmed to target any specific RNA or DNA sequence, these assays can be adapted for a wide range of applications. However, additional research and development is required to improve accuracy and speed further while reducing the cost of various CRISPR-based diagnostic assay systems.
CRISPR-Based Diagnostic Systems
One of the most well-known examples of CRISPR-based nucleic acid detection assays is the SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) system, developed by researchers at the Broad Institute of MIT and Harvard and commercialized by Sherlock Biosciences. The SHERLOCK system uses a CRISPR-Cas13a system to detect specific RNA or DNA sequences associated with diseases such as Zika, dengue fever, or COVID-191,2. The system is highly sensitive and specific, detecting as few as ten copies of the target sequence in a sample.
Another example of a CRISPR-based diagnostic assay is DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter), developed by researchers at UC Berkeley and commercialized by Mammoth Biosciences. DETECTR uses CRISPR-Cas12a system to detect specific DNA sequences associated with diseases such as HPV (human papillomavirus) and cancer3,4. The system can detect DNA mutations associated with cancer development, allowing doctors to identify and treat the disease in its early stages.
Additional CRISPR-based diagnostic systems under development include: HOLMES (DNA & RNA viruses)5, NASBACC (Zika)6, CRISPR-mediated DNA FISH system (MRSA)7, CAS-EXPAR (Listeria monocytogenes) 8, LEOPARD (SARS-CoV2)9, igRNA (HIV)10, and nCATS (cancer)11.
CRISPR-based diagnostic assays are also being developed for various other applications, including detecting foodborne pathogens and environmental pollutants. For example, researchers have developed a CRISPR-based biosensor that can detect the presence of E. coli in food samples12. The biosensor uses CRISPR to target specific DNA sequences associated with the bacteria, allowing food safety inspectors to quickly and accurately identify contaminated samples.
CRISPR gene editing is currently applied in crops and livestock, biofuel production, and invasive species or pest control to revolutionize agricultural biotechnology (AgBio). Though it is important to note that the use of CRISPR in agriculture is subject to various regulations and ethical considerations, it has already made significant impact in crop improvement, precision breeding, disease control, species conservation, etc. Here we examine how researchers apply transgene-free CRISPR RNP gene editing to address some global problems.
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 resistance1,2 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 yield4,5, 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 species18,19,20, 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 species22,23, 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.
