How CRISPR Edits Genes: A Molecular Guide for Students

By Dr. Zubair Khalid, DVM, MS, PhD ·

How CRISPR Edits Genes: A Molecular Guide for Students

Introduction to CRISPR Gene Editing

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a naturally occurring adaptive immune system found in bacteria and archaea. When a bacteriophage infects a bacterium, the host can capture a short fragment of the viral DNA and integrate it into its own genome at a CRISPR array locus. This stored sequence serves as a molecular memory: upon subsequent infection, the bacterium transcribes the stored spacer into a short RNA that guides a nuclease to cleave the incoming viral genome, destroying it before replication can proceed.

The revolutionary insight that transformed this bacterial defense system into a programmable gene-editing tool came from the work of Jennifer Doudna, Emmanuelle Charpentier, and colleagues, who demonstrated in 2012 that the Cas9 endonuclease could be reprogrammed with a synthetic guide RNA to cut any DNA sequence of interest in a test tube. The subsequent demonstration of CRISPR-mediated editing in human cells in 2013 opened the floodgates for its use across virtually every area of molecular biology.

This article provides a mechanistic account of how CRISPR edits genes, from the molecular components and the biochemistry of DNA cleavage to the cellular repair pathways that determine the editing outcome. It also covers guide RNA design principles, delivery methods, verification techniques, and common experimental pitfalls. By the end, you should be able to explain precisely what happens when you "use CRISPR" in the lab and why the outcome is never a single, simple event but rather a competition between distinct DNA repair pathways.

Key Components of the CRISPR System

The most widely used CRISPR system for gene editing is the type II CRISPR-Cas9 system from Streptococcus pyogenes. It requires two essential components: the Cas9 protein and a guide RNA. A third element, the PAM sequence, is not a component you introduce but a feature of the target DNA that determines where Cas9 can bind and cut.

Cas9 Protein

Cas9 is a large (approximately 160 kDa) multidomain endonuclease. It contains two nuclease domains: the HNH domain, which cleaves the DNA strand complementary to the guide RNA, and the RuvC domain, which cleaves the non-complementary strand. Both domains must be active for Cas9 to produce a double-strand break (DSB). If you mutate a critical catalytic residue in either domain—for example, D10A in RuvC or H840A in HNH—you generate a "nickase" version of Cas9 that cuts only one strand. Mutating both residues produces catalytically dead Cas9 (dCas9), which retains DNA-binding ability but cannot cleave. This dCas9 is the foundation of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies, discussed later.

Cas9 also contains two recognition lobes that undergo a large conformational change upon binding to the guide RNA and then to target DNA. In its apo state (without guide RNA), Cas9 is inactive. Binding to the guide RNA induces a structural rearrangement that primes the enzyme for DNA recognition.

Guide RNA (sgRNA)

In nature, the CRISPR system uses two RNA molecules: a CRISPR RNA (crRNA) that contains the spacer sequence complementary to the target, and a trans-activating crRNA (tracrRNA) that base-pairs with the crRNA and is required for Cas9 recruitment. In the laboratory, these two RNAs are fused into a single guide RNA (sgRNA) of approximately 100 nucleotides. The sgRNA has two functional regions:

  1. The 20-nucleotide spacer sequence at the 5′ end, which is complementary to the target DNA sequence. This is the region you design to match your gene of interest.
  2. The scaffold region, which forms a hairpin structure recognized by Cas9. This region is constant across all sgRNAs and is required for Cas9 binding.

The specificity of CRISPR-Cas9 is determined primarily by the 20-nucleotide spacer, but as discussed below, the PAM sequence imposes an additional constraint on where binding can occur.

PAM Sequence

The protospacer adjacent motif (PAM) is a short, conserved DNA sequence located immediately 3′ of the target site on the non-target strand. For S. pyogenes Cas9, the PAM is 5′-NGG-3′ (where N is any nucleotide). The PAM is essential for two reasons:

  • Initial DNA interrogation: Cas9 first scans the genome for PAM sequences. Binding to a PAM triggers local DNA unwinding, allowing the guide RNA to sample complementarity with the adjacent sequence. Without a PAM, Cas9 cannot initiate binding.
  • Self vs. non-self discrimination: The CRISPR array in the bacterial genome contains spacers that match the target, but the repeat sequences flanking them do not contain a PAM. This prevents Cas9 from cleaving the CRISPR array itself.

The PAM requirement means that not every 20-nucleotide sequence in a genome is targetable. For S. pyogenes Cas9, you need an NGG motif immediately downstream of your target site. Other Cas9 orthologs recognize different PAMs—for example, Staphylococcus aureus Cas9 recognizes 5′-NNGRRT-3′, and Cas12a (Cpf1) recognizes T-rich PAMs—expanding the range of targetable sequences.

Mechanism of CRISPR-Mediated Gene Editing

The process of CRISPR editing can be divided into two phases: the formation of a site-specific double-strand break and the subsequent repair of that break by the cell's endogenous DNA repair machinery. Understanding both phases is critical because the repair step, not the cleavage step, determines the final editing outcome.

Double-Strand Break Formation

The sequence of events leading to a DSB is as follows:

  1. R-loop formation: The Cas9-sgRNA complex binds to a PAM sequence in the target DNA. The PAM is recognized by the PAM-interacting domain of Cas9. Upon PAM binding, the DNA duplex is locally melted, and the guide RNA begins to base-pair with the complementary strand, forming an R-loop structure.
  2. Seed region hybridization: The first 8–12 nucleotides of the guide RNA adjacent to the PAM (the "seed region") are critical for specificity. Mismatches in this region severely impair cleavage, whereas mismatches in the distal region are more tolerated.
  3. Conformational activation: Full guide-target complementarity triggers a conformational change in Cas9 that aligns the HNH and RuvC nuclease domains with their respective DNA strands.
  4. Cleavage: The HNH domain cleaves the complementary strand, and the RuvC domain cleaves the non-complementary strand, producing a blunt double-strand break approximately 3 nucleotides upstream of the PAM.

The DSB is the initiating event for all downstream editing outcomes. Without a DSB, no editing occurs. This is why dCas9 (catalytically dead) cannot edit genes—it binds but does not cut.

Non-Homologous End Joining (NHEJ)

Once a DSB is formed, the cell must repair it or risk chromosome fragmentation and cell death. The dominant repair pathway in most mammalian cell types is non-homologous end joining (NHEJ). NHEJ is an error-prone process that directly ligates the two broken DNA ends together without requiring a homologous template.

The key steps of NHEJ are:

  1. End recognition: The Ku70/Ku80 heterodimer binds to the broken DNA ends.
  2. End processing: Nucleases (such as Artemis) and polymerases (such as DNA polymerase μ and λ) may trim or fill in the ends, creating small insertions or deletions (indels).
  3. Ligation: DNA ligase IV, in complex with XRCC4 and XLF, seals the ends.

Because NHEJ is error-prone, the repaired locus frequently contains indels. If the indel occurs within the coding sequence of a gene and introduces a frameshift or a premature stop codon, the result is a gene knockout. This is the basis of CRISPR Knockout experiments. The efficiency of NHEJ varies by cell type and locus but is typically the dominant outcome in actively dividing cells.

Homology-Directed Repair (HDR)

The alternative repair pathway is homology-directed repair (HDR), which uses a homologous DNA template to repair the break accurately. In the context of CRISPR editing, researchers can supply an exogenous donor template to exploit HDR for precise genome modifications, such as introducing a specific mutation or inserting a reporter gene.

HDR proceeds through the following steps:

  1. End resection: The DSB ends are resected by nucleases (such as MRE11 and CtIP) to generate 3′ single-stranded DNA overhangs.
  2. Strand invasion: The single-stranded overhang invades a homologous duplex DNA template, forming a displacement loop (D-loop).
  3. DNA synthesis: The invading strand is extended by DNA polymerases using the template.
  4. Resolution: The resulting Holliday junctions are resolved, and the break is repaired.

For CRISPR-mediated HDR, the donor template can be delivered as:

  • Single-stranded oligodeoxynucleotides (ssODNs): Typically 100–200 nucleotides long, with homology arms of 40–60 nucleotides flanking the desired edit. These are used for introducing small mutations or short insertions.
  • Plasmid donor vectors: Containing longer homology arms (typically 500–1000 nucleotides each) flanking a selection cassette or a larger insert.

HDR is generally less efficient than NHEJ in most cell types, particularly in non-dividing cells, because HDR is largely restricted to the S/G2 phases of the cell cycle when sister chromatids are available as templates. Strategies to enhance HDR efficiency include synchronizing cells in S phase, using small-molecule inhibitors of NHEJ (such as SCR7), or using Cas9 variants with modified repair pathway preferences.

The choice between NHEJ and HDR is a central consideration in experimental design. If you want to knock out a gene, NHEJ is sufficient and often desirable. If you want to introduce a precise mutation or a fluorescent tag, you must design an HDR donor and optimize conditions to favor HDR over NHEJ.

Designing Guide RNAs for Specific Gene Targeting

The success of a CRISPR experiment hinges on the design of the sgRNA. A poorly designed sgRNA can result in no editing, off-target cleavage, or both. Several principles guide sgRNA design.

Target Site Selection

The first step is to identify a 20-nucleotide sequence in your gene of interest that is immediately followed by an NGG PAM. The target sequence should be:

  • Unique in the genome: Use a BLAST search or a dedicated sgRNA design tool (such as Benchling, CRISPOR, or CHOPCHOP) to ensure the sequence does not have significant homology elsewhere in the genome.
  • Located in a critical region of the gene: For knockout experiments, target the earliest possible exon that is common to all transcript isoforms, ideally within the first 5% of the coding sequence. This maximizes the chance that an indel will produce a truncated, non-functional protein. Targeting the 3′ end of the gene may produce a partially functional protein if the truncation is small.
  • Avoiding secondary structure: The sgRNA should not form stable secondary structures that could interfere with Cas9 loading or target recognition.

For HDR experiments, the cut site should be as close as possible to the intended edit. The efficiency of HDR drops sharply with distance from the DSB; edits more than 30 nucleotides from the cut site are often inefficient.

Off-Target Effects

Off-target effects occur when Cas9 cleaves at genomic sites that are similar but not identical to the target sequence. The seed region (positions 1–12 adjacent to the PAM) is most critical for specificity, but mismatches in the PAM-distal region are tolerated to varying degrees. The most common off-target sites are those with a few mismatches in the PAM-distal region and a canonical NGG PAM.

Strategies to minimize off-target effects include:

  • Using high-fidelity Cas9 variants: Engineered variants such as SpCas9-HF1 and eSpCas9 have reduced off-target activity while maintaining on-target efficiency.
  • Shortening the guide RNA: Truncated sgRNAs (17–18 nucleotides) can reduce off-target effects, though they may also reduce on-target efficiency.
  • Using paired nickases: Two Cas9 nickases targeting adjacent sites on opposite strands can create a DSB with high specificity, as off-target nicks are repaired without producing a DSB.
  • Selecting target sites with minimal homology to other genomic loci: Design tools provide off-target scores that predict the likelihood of off-target cleavage.

Delivery Methods for CRISPR Components

To edit a cell, you must deliver the Cas9 protein and the guide RNA (or the DNA encoding them) into the nucleus. The choice of delivery method depends on the cell type, the experimental goal, and whether you need transient or stable expression.

Plasmid-Based Delivery

The most common approach in academic labs is to transfect a plasmid that encodes both Cas9 and the sgRNA. Plasmids are relatively inexpensive and easy to construct or obtain from repositories such as Addgene. However, plasmid delivery has several limitations:

  • Expression is transient but prolonged: Plasmid-encoded Cas9 can persist for days, increasing the risk of off-target effects.
  • Transfection efficiency varies: Some cell types (e.g., primary cells, neurons) are difficult to transfect with standard lipid-based reagents.
  • Potential for random integration: Although rare, plasmid DNA can integrate into the genome, which is a concern for therapeutic applications.

Viral Vectors

For hard-to-transfect cells or for in vivo delivery, viral vectors are often used. Adeno-associated virus (AAV) is the most popular choice because it is non-pathogenic, has a low immunogenicity, and can transduce both dividing and non-dividing cells. However, AAV has a limited packaging capacity (~4.7 kb), which is a constraint because SpCas9 alone is ~4.2 kb. This limitation has driven the development of smaller Cas9 orthologs, such as S. aureus Cas9 (SaCas9), which is ~3.2 kb and can be packaged alongside an sgRNA in a single AAV vector.

Lentiviral vectors can package larger inserts and integrate into the genome, providing stable expression. However, integration carries the risk of insertional mutagenesis and is generally avoided for therapeutic applications.

Ribonucleoprotein (RNP) Delivery

The RNP approach involves delivering the Cas9 protein pre-complexed with the sgRNA directly into cells. This is achieved by electroporation or by using lipid-based transfection reagents that deliver proteins. RNP delivery has several advantages:

  • Rapid onset of activity: Since the protein is already present, editing begins immediately after delivery.
  • Transient activity: The protein is degraded within 24–72 hours, reducing off-target effects.
  • No DNA integration risk: There is no plasmid or viral DNA that could integrate into the genome.
  • Lower immunogenicity: For therapeutic applications, RNPs avoid the risk of persistent Cas9 expression that could trigger an immune response.

The main disadvantage of RNP delivery is the cost of producing recombinant Cas9 protein and the need for electroporation equipment, which can be harsh on cells.

Applications of CRISPR in Research and Medicine

The ability to create targeted DSBs and exploit repair pathways has enabled a wide range of applications, from basic research to clinical therapy.

Gene Knockout and Knock-in

The most straightforward application is gene knockout via NHEJ-induced indels. By targeting the first coding exon of a gene, you can generate frameshift mutations that abolish protein function. This is the foundation of CRISPR Knockout screens, where libraries of sgRNAs targeting thousands of genes are used to identify genes essential for a phenotype of interest.

Knock-in experiments use HDR to introduce precise modifications, such as point mutations, epitope tags, or fluorescent reporters. For example, you can fuse GFP to an endogenous gene by providing a donor template with homology arms flanking the insertion site. This approach has been used extensively to study protein localization and dynamics in living cells.

CRISPRi and CRISPRa

Catalytically dead Cas9 (dCas9) retains the ability to bind DNA but cannot cleave it. When dCas9 is fused to transcriptional repressors (such as KRAB) or activators (such as VP64), it can be targeted to gene promoters to repress or activate transcription, respectively. These technologies are known as CRISPR Knock (CRISPR interference) and CRISPR activation (CRISPRa).

CRISPRi and CRISPRa are powerful tools for studying gene function without altering the underlying DNA sequence. They are reversible, titratable, and can be used to modulate the expression of multiple genes simultaneously. CRISPRi is particularly useful for essential genes, where complete knockout would be lethal.

Clinical Trials

CRISPR has entered the clinic for several applications. The most advanced is the treatment of sickle cell disease and beta-thalassemia using ex vivo editing of hematopoietic stem cells. In this approach, patient-derived stem cells are edited to reactivate fetal hemoglobin expression, then re-infused into the patient. The FDA approved the first CRISPR-based therapy, Casgevy (exagamglogene autotemcel), in 2023 for sickle cell disease.

Other clinical applications include CAR-T cell therapies where CRISPR is used to knock out genes that inhibit T-cell function, and in vivo editing for conditions such as transthyretin amyloidosis, where CRISPR is delivered to the liver to knock out the disease-causing gene. These efforts are reviewed in more detail under CRISPR in Medicine.

Methods to Detect and Verify Gene Editing

After performing a CRISPR experiment, you must verify that editing occurred and characterize the nature of the edits. Several complementary methods are available.

Surveyor/T7E1 Assay

The T7 endonuclease I (T7E1) assay is a rapid, inexpensive method to detect the presence of indels in a mixed cell population. The principle is as follows:

  1. PCR amplification: Amplify the target region by PCR from genomic DNA extracted from edited cells.
  2. Denaturation and reannealing: Heat the PCR product to denature the duplex, then slowly cool to allow reannealing. If indels are present, some duplexes will contain mismatches where a wild-type strand anneals to a mutant strand.
  3. T7E1 digestion: T7E1 recognizes and cleaves mismatched DNA. The digestion products are resolved by agarose gel electrophoresis.
  4. Quantification: The intensity of the cleavage bands relative to the full-length product provides an estimate of editing efficiency.

The T7E1 assay is simple but has limitations: it underestimates editing efficiency, cannot distinguish between different indel types, and is not quantitative for homozygous vs. heterozygous edits.

Sanger Sequencing

Sanger sequencing of the target locus provides more detailed information. If you sequence PCR products from a mixed population, the chromatogram will show overlapping peaks downstream of the cut site, indicating the presence of multiple alleles. This can be analyzed using tools such as TIDE (Tracking of Indels by Decomposition) or ICE (Inference of CRISPR Edits), which deconvolute the sequencing trace to estimate the frequency and types of indels.

For precise characterization, you can clone individual PCR products into plasmids and sequence individual colonies. This provides the exact sequence of each edited allele but is labor-intensive.

For HDR experiments, you should also verify that the desired edit was introduced and that no unintended mutations are present. This typically requires Sanger sequencing of the entire homology arm region.

Common Pitfalls and How to Avoid Them

Even experienced researchers encounter failures in CRISPR experiments. Understanding the most common pitfalls can save you significant time and frustration.

Off-Target Effects

Off-target cleavage is a major concern, particularly for therapeutic applications. Even with careful sgRNA design, off-target effects can occur at sites with several mismatches. To mitigate this:

  • Always use the most specific sgRNA design tools and check off-target scores.
  • Consider using high-fidelity Cas9 variants or paired nickases.
  • Validate editing at predicted off-target sites by PCR and sequencing.
  • For critical experiments, use unbiased methods such as whole-genome sequencing to assess off-target effects.

Incorrect PAM Usage

A common mistake is designing an sgRNA without verifying the PAM sequence. For S. pyogenes Cas9, the PAM must be 5′-NGG-3′ immediately downstream of the target. If you use a different Cas9 ortholog, the PAM requirement changes. Always confirm that your target site has a valid PAM and that the PAM is on the correct strand.

Misinterpreting NHEJ vs HDR

Students often expect that CRISPR editing will produce the desired precise edit. In reality, NHEJ is the dominant repair pathway in most cells, and HDR is inefficient. If you are attempting a knock-in, you must:

  • Design a donor template with appropriate homology arms.
  • Optimize delivery to maximize HDR (e.g., using ssODNs for small edits).
  • Use selection or enrichment strategies (e.g., fluorescent reporters or antibiotic resistance markers) to isolate successfully edited cells.
  • Be aware that even in successfully edited cells, the other allele may have undergone NHEJ.

Summary and Key Takeaways

CRISPR gene editing is a powerful technology that harnesses a bacterial immune system to introduce targeted double-strand breaks in DNA, which are then repaired by the cell's endogenous pathways. The outcome of editing is determined by the competition between NHEJ and HDR, not by the Cas9 cleavage itself.

  • CRISPR-Cas9 requires two components: the Cas9 endonuclease and a single guide RNA (sgRNA) with a 20-nucleotide spacer that determines target specificity.
  • The PAM sequence (5′-NGG-3′ for SpCas9) is essential for Cas9 binding and cleavage; without it, no editing occurs.
  • NHEJ is the dominant repair pathway and produces indels, which can be used for gene knockout.
  • HDR uses a donor template and enables precise edits, but is less efficient and requires optimization.
  • Guide RNA design is critical: target early exons, avoid off-target homology, and verify PAM availability.
  • Delivery methods include plasmids, viral vectors, and ribonucleoprotein complexes, each with distinct advantages and limitations.
  • Verification of editing requires PCR-based assays (T7E1), Sanger sequencing, and, for precise edits, cloning and sequencing of individual alleles.

Frequently Asked Questions

How does CRISPR edit genes?

CRISPR edits genes by introducing a targeted double-strand break (DSB) at a specific genomic location. The Cas9 protein, guided by a synthetic RNA molecule, binds to a 20-nucleotide target sequence adjacent to a PAM motif and cleaves both DNA strands. The cell then repairs the DSB using either non-homologous end joining (NHEJ), which creates insertions or deletions (indels) that can disrupt gene function, or homology-directed repair (HDR), which uses a donor template to introduce precise sequence changes. The repair pathway, not the cleavage event, determines the final editing outcome.

What is the role of the PAM sequence in CRISPR?

The protospacer adjacent motif (PAM) is a short DNA sequence (5′-NGG-3′ for S. pyogenes Cas9) located immediately downstream of the target site. The PAM is required for Cas9 to initially bind and interrogate the DNA. Cas9 scans the genome for PAM sequences; only when a PAM is bound does Cas9 unwind the adjacent DNA and allow the guide RNA to test complementarity. The PAM also prevents Cas9 from cleaving the CRISPR array itself in bacteria, as the repeat sequences lack a PAM.

What is the difference between NHEJ and HDR?

NHEJ (non-homologous end joining) is an error-prone repair pathway that directly ligates broken DNA ends, often introducing small insertions or deletions (indels). It is active throughout the cell cycle and is the dominant repair pathway in most cells. HDR (homology-directed repair) uses a homologous DNA template to repair the break accurately and is restricted to the S/G2 phases of the cell cycle. In CRISPR experiments, NHEJ is used for gene knockout, while HDR is used for precise edits such as point mutations or gene insertions.

How do you design a guide RNA for a specific gene?

To design a guide RNA, you first identify a 20-nucleotide sequence in your gene of interest that is immediately followed by an NGG PAM. The sequence should be unique in the genome to minimize off-target effects, and it should be located in a critical region of the gene—typically the first coding exon for knockout experiments. Use design tools such as CRISPOR, CHOPCHOP, or Benchling, which provide on-target and off-target scores to help select the best guide.

What are off-target effects in CRISPR?

Off-target effects are Cas9-mediated cleavage events at genomic sites that are similar but not identical to the intended target sequence. These occur because the guide RNA can tolerate mismatches, particularly in the PAM-distal region. Off-target cleavage can cause unintended mutations, chromosomal rearrangements, or cell death. Strategies to reduce off-target effects include using high-fidelity Cas9 variants, truncated guide RNAs, paired nickases, and careful sgRNA design with computational off-target prediction.

How is CRISPR delivered into cells?

CRISPR components can be delivered as DNA plasmids encoding Cas9 and the sgRNA, as viral vectors (such as AAV or lentivirus), or as ribonucleoprotein (RNP) complexes where the Cas9 protein is pre-complexed with the sgRNA and delivered directly. Plasmid delivery is common for easy-to-transfect cells, viral vectors are used for hard-to-transfect cells or in vivo delivery, and RNP delivery offers rapid, transient activity with reduced off-target effects.

What is CRISPRi and CRISPRa?

CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) use a catalytically dead Cas9 (dCas9) that can bind DNA but cannot cleave it. When dCas9 is fused to a transcriptional repressor (such as KRAB), it can be targeted to gene promoters to silence gene expression (CRISPRi). When fused to transcriptional activators (such as VP64), it can upregulate gene expression (CRISPRa). These tools allow reversible, tunable control of gene expression without altering the underlying DNA sequence.

How do you verify that CRISPR editing worked?

Editing is typically verified by PCR amplification of the target region followed by the T7E1 mismatch cleavage assay, which detects the presence of indels. Sanger sequencing of the PCR product provides more detailed information about the types and frequencies of edits, especially when analyzed with tools like TIDE or ICE. For precise HDR edits, cloning and sequencing individual alleles is recommended to confirm the exact sequence change.

Key Takeaways

  • CRISPR-Cas9 is a programmable nuclease that introduces targeted double-strand breaks; the repair outcome (NHEJ vs. HDR) determines whether you get a knockout or a precise edit.
  • The PAM sequence is essential for Cas9 function; always verify PAM availability when designing guide RNAs.
  • Guide RNA design is the most critical determinant of editing success and specificity; use computational tools and validate off-target sites.
  • NHEJ is efficient but error-prone; HDR is precise but inefficient, requiring careful optimization of donor templates and delivery.
  • Delivery method choice (plasmid, viral, RNP) depends on cell type, experimental goal, and the need for transient vs. stable expression.
  • Verification of editing requires multiple approaches, including T7E1 assays, Sanger sequencing, and, for precise edits, clonal sequencing.
  • CRISPR is not just a gene-editing tool; dCas9-based technologies (CRISPRi/a) enable transcriptional control, and clinical applications are rapidly expanding.

Further Reading

  • Song B, Bae S. Genome editing using CRISPR, CAST, and Fanzor systems. Molecules and cells. 2024. PubMed 38909984
  • Babačić H et al. CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: A systematic review. PloS one. 2019. PubMed 30794581
  • Liu C et al. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. Journal of controlled release : official journal of the Controlled Release Society. 2017. PubMed 28911805
  • Hodges CA, Conlon RA. Delivering on the promise of gene editing for cystic fibrosis. Genes & diseases. 2019. PubMed 31193992
  • Nazir R et al. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genome-editing toolkit to enhance salt stress tolerance in rice and wheat. Physiologia plantarum. 2022. PubMed 35099818
  • McDonnell L et al. CRISPR in Your Kitchen: an At-Home CRISPR Kit to Edit Genes in Saccharomyces cerevisiae Used during a Remote Lab Course. Journal of microbiology & biology education. 2022. PubMed 35496692

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