CRISPR gene editing

Part of a series on
CRISPR
Genome editing CRISPR gene editing
Variants
Anti-CRISPR CIRTS CRISPeY CRISPR-Cas10 CRISPR-Cas13 CRISPR-BEST CRISPR-Disp CRISPR-Gold CRISPRa CRISPRi Easi-CRISPR FACE
Enzyme
Cas9 FokI EcoRI PstI SmaI HaeIII Cas12a (Cpf1) xCas9
Applications
CAMERA ICE Genética dirigida
Other genome editing methods
Prime editing Pro-AG RESCUE TALEN ZFN LEAPER
v t e

CRISPR gene editing (/ˈkrɪspər/; pronounced like "crisper"; an abbreviation for "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed or new ones added in vivo.^[1]

The technique is considered highly significant in biotechnology and medicine as it enables editing genomes in vivo and is precise, cost-effective, and efficient. It can be used in the creation of new medicines, agricultural products, and genetically modified organisms, or as a means of controlling pathogens and pests. It also offers potential in the treatment of inherited genetic diseases as well as diseases arising from somatic mutations such as cancer. However, its use in human germline genetic modification is highly controversial. The development of this technique earned Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize in Chemistry in 2020.^[2][3] The third researcher group that shared the Kavli Prize for the same discovery,^[4] led by Virginijus Šikšnys, was not awarded the Nobel prize.^[5][6][7]

Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via homology directed repair (HDR), is the traditional pathway of targeted genomic editing approaches.^[1] This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template.^[1] This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for the repair to commence. Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality. Therefore, genomic engineering by CRISPR-Cas9 gives researchers the ability to generate targeted random gene disruption.

While genome editing in eukaryotic cells has been possible using various methods since the 1980s, the methods employed had proven to be inefficient and impractical to implement on a large scale. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing became possible. Cas9 derived from the bacterial species Streptococcus pyogenes has facilitated targeted genomic modification in eukaryotic cells by allowing for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRNA guide strands.^[8] Researchers can insert Cas9 and template RNA with ease in order to silence or cause point mutations at specific loci. This has proven invaluable for quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. Newly engineered variants of the Cas9 nuclease that significantly reduce off-target activity have been developed.^[9]

CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing^[10] was the AAAS's choice for Breakthrough of the Year in 2015.^[11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos.^[12] In 2023, the first drug making use of CRISPR gene editing, Casgevy, was approved for use in the United Kingdom, to cure sickle-cell disease and beta thalassemia.^[13][14]. On 2 December 2023, the Kingdom of Bahrain became the second country in the world to approve the use of Casgevy, to treat sickle-cell anemia and beta thalassemia.^[15][16] Casgevy was approved for use in the United States on December 8, 2023, by the Food and Drug Administration.^[17]

In the early 2000s, German researchers began developing zinc finger nucleases (ZFNs), synthetic proteins whose DNA-binding domains enable them to create double-stranded breaks in DNA at specific points. ZFNs have a higher precision and the advantage of being smaller than Cas9, but ZFNs are not as commonly used as CRISPR-based methods. In 2010, synthetic nucleases called transcription activator-like effector nucleases (TALENs) provided an easier way to target a double-stranded break to a specific location on the DNA strand. Both zinc finger nucleases and TALENs require the design and creation of a custom protein for each targeted DNA sequence, which is a much more difficult and time-consuming process than that of designing guide RNAs. CRISPRs are much easier to design because the process requires synthesizing only a short RNA sequence, a procedure that is already widely used for many other molecular biology techniques (e.g. creating oligonucleotide primers).^[18]

Whereas methods such as RNA interference (RNAi) do not fully suppress gene function, CRISPR, ZFNs, and TALENs provide full, irreversible gene knockout.^[19] CRISPR can also target several DNA sites simultaneously simply by introducing different gRNAs. In addition, the costs of employing CRISPR are relatively low.^[19][20][21]

In 2005, Alexander Bolotin at the French National Institute for Agricultural Research (INRA) discovered a CRISPR locus that contained novel Cas genes, significantly one that encoded a large protein known as Cas9.^[22]

In 2006, Eugene Koonin at the US National Center for Biotechnology information, NIH, proposed an explanation as to how CRISPR cascades as a bacterial immune system.^[22]

In 2007, Philippe Horvath at Danisco France SAS displayed experimentally how CRISPR systems are an adaptive immune system, and integrate new phage DNA into the CRISPR array, which is how they fight off the next wave of attacking phage.^[22]

In 2012, the research team led by professor Jennifer Doudna (University of California, Berkeley) and professor Emmanuelle Charpentier (Umeå University) were the first people to identify, disclose, and file a patent application for the CRISPR-Cas9 system needed to edit DNA.^[22] They also published their finding that CRISPR-Cas9 could be programmed with RNA to edit genomic DNA, now considered one of the most significant discoveries in the history of biology.

As of November 2013^[update], SAGE Labs (part of Horizon Discovery group) had exclusive rights from one of those companies to produce and sell genetically engineered rats and non-exclusive rights for mouse and rabbit models.^[23] By 2015^[update], Thermo Fisher Scientific had licensed intellectual property from ToolGen to develop CRISPR reagent kits.^[24]

As of December 2014^[update], patent rights to CRISPR were contested. Several companies formed to develop related drugs and research tools.^[25] As companies ramped up financing, doubts as to whether CRISPR could be quickly monetized were raised.^[26] In 2014, Feng Zhang of the Broad Institute of MIT and Harvard and nine others were awarded US patent number 8,697,359^[27] over the use of CRISPR–Cas9 gene editing in eukaryotes. Although Charpentier and Doudna (referred to as CVC) were credited for the conception of CRISPR, the Broad Institute was the first to achieve a "reduction to practice" according to patent judges Sally Gardner Lane, James T. Moore and Deborah Katz.^[28]

The first set of patents was awarded to the Broad team in 2015, prompting attorneys for the CVC group to request the first interference proceeding.^[29] In February 2017, the US Patent Office ruled on a patent interference case brought by University of California with respect to patents issued to the Broad Institute, and found that the Broad patents, with claims covering the application of CRISPR-Cas9 in eukaryotic cells, were distinct from the inventions claimed by University of California.^[30][31][32]

Shortly after, University of California filed an appeal of this ruling.^[33][34] In 2019 the second interference dispute was opened. This was in response to patent applications made by CVC that required the appeals board to determine the original inventor of the technology. The USPTO ruled in March 2022 against UC, stating that the Broad Institute were first to file. The decision affected many of the licensing agreements for the CRISPR editing technology that was licensed from UC Berkeley. UC stated its intent to appeal the USPTO's ruling.^[35]

In March 2017, the European Patent Office (EPO) announced its intention to allow claims for editing all types of cells to Max-Planck Institute in Berlin, University of California, and University of Vienna,^[36][37] and in August 2017, the EPO announced its intention to allow CRISPR claims in a patent application that MilliporeSigma had filed.^[36] As of August 2017^[update] the patent situation in Europe was complex, with MilliporeSigma, ToolGen, Vilnius University, and Harvard contending for claims, along with University of California and Broad.^[38]

In July 2018, the ECJ ruled that gene editing for plants was a sub-category of GMO foods and therefore that the CRISPR technique would henceforth be regulated in the European Union by their rules and regulations for GMOs.^[39]

In February 2020, a US trial showed safe CRISPR gene editing on three cancer patients.^[40]

In October 2020, researchers Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their work in this field.^[41][42] They made history as the first two women to share this award without a male contributor.^[43][5]

In June 2021, the first, small clinical trial of intravenous CRISPR gene editing in humans concluded with promising results.^[44][45]

In September 2021, the first CRISPR-edited food went on public sale in Japan. Tomatoes were genetically modified for around five times the normal amount of possibly calming^[46] GABA.^[47] CRISPR was first applied in tomatoes in 2014.^[48]

In December 2021, it was reported that the first CRISPR-gene-edited marine animal/seafood and second set of CRISPR-edited food has gone on public sale in Japan: two fish of which one species grows to twice the size of natural specimens due to disruption of leptin, which controls appetite, and the other grows to 1.2 times the natural average size with the same amount of food due to disabled myostatin, which inhibits muscle growth.^[49][50][51]

A 2022 study has found that knowing more about CRISPR tomatoes had a strong effect on the participants' preference. "Almost half of the 32 participants from Germany who are scientists demonstrated constant choices, while the majority showed increased willingness to buy CRISPR tomatoes, mostly non-scientists."^[52][53]

In May 2021, UC Berkeley announced their intent to auction non-fungible tokens of both the patent for CRISPR gene editing as well as cancer immunotherapy. However, the university would in this case retain ownership of the patents.^[54][55] 85 % of funds gathered through the sale of the collection named The Fourth Pillar were to be used to finance research.^[56][57] It sold in June 2022 for 22 Ether, which was around US$54,000 at the time.^[58]

In November 2023, the United Kingdom's Medicines and Healthcare products Regulatory Agency (MHRA) became the first in the world to approve the use of the first drug based on CRISPR gene editing, Casgevy, to treat sickle-cell anemia and beta thalassemia. Casgevy, or exagamglogene autotemcel, directly acts on the genes of the stem cells inside the patient's bones, having them produce healthy red blood cells. This treatment thus avoids the need for regular, costly blood transfusions.^[13][14]

On 2 December 2023, the Kingdom of Bahrain's National Health Regulatory Authority (NHRA) became the second in the world to approve the use of Casgevy, to treat sickle-cell anemia and beta thalassemia.^[59][60]

In December 2023, the FDA approved the first gene therapy in the US to treat patients with Sickle Cell Disease (SCD). The FDA approved two milestone treatments, Casgevy and Lyfgenia, representing the first cell-based gene therapies for the treatment of SCD.^[61]

On 16 February 2025, the Kingdom of Bahrain's announced the successful completion of treatment with Casgevy for the world's first sickle cell disease patient outside of the United States at the Royal Medical Services- Bahrain Oncology Center, located at King Hamad University Hospital (KHUH)^[62][63], and was congratulated by the World Health Organisation's Director General, Tedros Adhanom Ghebreyesus for the successful administration of the treatment.^[64]

CRISPR-Cas9 genome editing uses a Type II CRISPR system. This system includes a ribonucleoprotein (RNP), consisting of Cas9, crRNA, and tracrRNA, along with an optional DNA repair template.

CRISPR-Cas9 often employs plasmids that code for the RNP components to transfect the target cells, or the RNP is assembled before addition to the cells via nucleofection.^[65] The main components of this plasmid are displayed in the image and listed in the table. The crRNA is uniquely designed for each application, as this is the sequence that Cas9 uses to identify and directly bind to specific sequences within the host cell's DNA. The crRNA must bind only where editing is desired. The repair template is also uniquely designed for each application, as it must complement to some degree the DNA sequences on either side of the cut and also contain whatever sequence is desired for insertion into the host genome.

Multiple crRNAs and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA).^[66] This sgRNA can be included alongside the gene that codes for the Cas9 protein and made into a plasmid in order to be transfected into cells. Many online tools are available to aid in designing effective sgRNA sequences.^[67][68]

Alternative proteins to Cas9 include the following:

CRISPR-Cas9 offers a high degree of fidelity and relatively simple construction. It depends on two factors for its specificity: the target sequence and the protospacer adjacent motif (PAM) sequence. The target sequence is 20 bases long as part of each CRISPR locus in the crRNA array.^[65] A typical crRNA array has multiple unique target sequences. Cas9 proteins select the correct location on the host's genome by utilizing the sequence to bond with base pairs on the host DNA. The sequence is not part of the Cas9 protein and as a result is customizable and can be independently synthesized.^[82][83]

The PAM sequence on the host genome is recognized by Cas9. Cas9 cannot be easily modified to recognize a different PAM sequence. However, this is ultimately not too limiting, as it is typically a very short and nonspecific sequence that occurs frequently at many places throughout the genome (e.g. the SpCas9 PAM sequence is 5'-NGG-3' and in the human genome occurs roughly every 8 to 12 base pairs).^[65]

Once these sequences have been assembled into a plasmid and transfected into cells, the Cas9 protein with the help of the crRNA finds the correct sequence in the host cell's DNA and – depending on the Cas9 variant – creates a single- or double-stranded break at the appropriate location in the DNA.^[84]

Properly spaced single-stranded breaks in the host DNA can trigger homology directed repair, which is less error-prone than the non-homologous end joining or theta-mediated end joining that typically follows a double-stranded break. Providing a DNA repair template allows for the insertion of a specific DNA sequence at an exact location within the genome. The repair template should extend 40 to 90 base pairs beyond the Cas9-induced DNA break.^[65] The goal is for the cell's native HDR process to utilize the provided repair template and thereby incorporate the new sequence into the genome. Once incorporated, this new sequence is now part of the cell's genetic material and passes into its daughter cells. Combined transient inhibition of NHEJ and TMEJ by a small molecule and siRNAs can increase HDR efficiency to up to 93% and simultaneously prevent off-target editing.^[85]

Delivery of Cas9, sgRNA, and associated complexes into cells can occur via viral and non-viral systems. Electroporation of DNA, RNA, or ribonucleocomplexes is a common technique, though it can result in harmful effects on the target cells.^[86] Chemical transfection techniques utilizing lipids and peptides have also been used to introduce sgRNAs in complex with Cas9 into cells.^[87][88] Nanoparticle-based delivery has also been used for transfection.^[89] Types of cells that are more difficult to transfect (e.g., stem cells, neurons, and hematopoietic cells) require more efficient delivery systems, such as those based on lentivirus (LVs), adenovirus (AdV), and adeno-associated virus (AAV).^[90][91][92]

Efficiency of CRISPR-Cas9 has been found to greatly increase when various components of the system including the entire CRISPR/Cas9 structure to Cas9-gRNA complexes delivered in assembled form rather than using transgenics.^[93][94] This has found particular value in genetically modified crops for mass commercialization.^[95][96] Since the host's replication machinery is not needed to produce these proteins, the chance of the recognizing sequence of the sgRNA is almost none, decreasing the chance of off-target effects.^[89]

Further improvements and variants of the CRISPR-Cas9 system have focused on introducing more control into its use. Specifically, the research aimed at improving this system includes improving its specificity, its efficiency, and the granularity of its editing power. Techniques can further be divided and classified by the component of the system they modify. These include using different variants or novel creations of the Cas protein, using an altogether different effector protein, modifying the sgRNA, or using an algorithmic approach to identify existing optimal solutions.

Specificity is an important aspect to improve the CRISPR-Cas9 system because the off-target effects it generates have serious consequences for the genome of the cell and invokes caution for its use. Minimizing off-target effects is thus maximizing the safety of the system. Novel variations of Cas9 proteins that increase specificity include effector proteins with comparable efficiency and specificity to the original SpCas9 that are able to target the previously untargetable sequences and a variant that has virtually no off-target mutations.^[97][98] Research has also been conducted in engineering new Cas9 proteins, including some that partially replace RNA nucleotides in crRNA with DNA and a structure-guided Cas9 mutant generating procedure that all had reduced off-target effects.^[99][100] Iteratively truncated sgRNAs and highly stabilized gRNAs have been shown to also decrease off-target effects.^[101][102] Computational methods including machine learning have been used to predict the affinity of and create unique sequences for the system to maximize specificity for given targets.^[103][104]

Several variants of CRISPR-Cas9 allow gene activation or genome editing with an external trigger such as light or small molecules.^{[105][106][107]} These include photoactivatable CRISPR systems developed by fusing light-responsive protein partners with an activator domain and a dCas9 for gene activation,^[108][109] or by fusing similar light-responsive domains with two constructs of split-Cas9,^[110][111] or by incorporating caged unnatural amino acids into Cas9,^[112] or by modifying the guide RNAs with photocleavable complements for genome editing.^[113]

Methods to control genome editing with small molecules include an allosteric Cas9, with no detectable background editing, that will activate binding and cleavage upon the addition of 4-hydroxytamoxifen (4-HT),^[105] 4-HT responsive intein-linked Cas9,^[114] or a Cas9 that is 4-HT responsive when fused to four ERT2 domains.^[115] Intein-inducible split-Cas9 allows dimerization of Cas9 fragments^[116] and rapamycin-inducible split-Cas9 system developed by fusing two constructs of split-Cas9 with FRB and FKBP fragments.^[117] Other studies have been able to induce transcription of Cas9 with a small molecule, doxycycline.^[118][119] Small molecules can also be used to improve homology directed repair,^[120] often by inhibiting the non-homologous end joining pathway and/or the theta-mediated end-joining pathway.^[121][122] A system with the Cpf1 effector protein was created that is induced by small molecules VE-822 and AZD-7762.^[123] These systems allow conditional control of CRISPR activity for improved precision, efficiency, and spatiotemporal control. Spatiotemporal control is a form of removing off-target effects—only certain cells or parts of the organism may need to be modified, and thus light or small molecules can be used as a way to conduct this. Efficiency of the CRISPR-Cas9 system is also greatly increased by proper delivery of the DNA instructions for creating the proteins and necessary reagents.^[123]

CRISPR also utilizes single base-pair editing proteins to create specific edits at one or two bases in the target sequence. CRISPR/Cas9 was fused with specific enzymes that initially could only change C to T and G to A mutations and their reverse. This was accomplished eventually without requiring any DNA cleavage.^{[124][125][126]} With the fusion of another enzyme, the base editing CRISPR-Cas9 system can also edit C to G and its reverse.^[127]

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a gene-editing technology that can induce double-strand breaks (DSBs) anywhere guide ribonucleic acids (gRNA) can bind with the protospacer adjacent motif (PAM) sequence.^[128] Single-strand nicks can also be induced by Cas9 active-site mutants,^[129] also known as Cas9 nickases.^[130] By simply changing the sequence of gRNA, the Cas9-endonuclease can be delivered to a gene of interest and induce DSBs.^[131] The efficiency of Cas9-endonuclease and the ease by which genes can be targeted led to the development of CRISPR-knockout (KO) libraries both for mouse and human cells, which can cover either specific gene sets of interest or the whole-genome.^[132][133] CRISPR screening helps scientists to create a systematic and high-throughput genetic perturbation within live model organisms. This genetic perturbation is necessary for fully understanding gene function and epigenetic regulation.^[134] The advantage of pooled CRISPR libraries is that more genes can be targeted at once.^{[citation needed]}

Knock-out libraries are created in a way to achieve equal representation and performance across all expressed gRNAs and carry an antibiotic or fluorescent selection marker that can be used to recover transduced cells.^[128] There are two plasmid systems in CRISPR/Cas9 libraries. First, is all in one plasmid, where sgRNA and Cas9 are produced simultaneously in a transfected cell. Second, is a two-vector system: sgRNA and Cas9 plasmids are delivered separately.^[134] It is important to deliver thousands of unique sgRNAs-containing vectors to a single vessel of cells by viral transduction at low multiplicity of infection (MOI, typically at 0.1–0.6), it prevents the probability that an individual cell clone will get more than one type of sgRNA otherwise it can lead to incorrect assignment of genotype to phenotype.^[132]

Once a pooled library is prepared it is necessary to carry out a deep sequencing (NGS, next generation sequencing) of PCR-amplified plasmid DNA in order to reveal abundance of sgRNAs. Cells of interest can be consequentially infected by the library and then selected according to the phenotype. There are 2 types of selection: negative and positive. By negative selection dead or slow growing cells are efficiently detected. It can identify survival-essential genes, which can further serve as candidates for molecularly targeted drugs. On the other hand, positive selection gives a collection of growth-advantage acquired populations by random mutagenesis.^[128] After selection genomic DNA is collected and sequenced by NGS. Depletion or enrichment of sgRNAs is detected and compared to the original sgRNA library, annotated with the target gene that sgRNA corresponds to. Statistical analysis then identifies genes that are significantly likely to be relevant to the phenotype of interest.^[132]

Apart from knock-out there are also knock-down (CRISPRi) and activation (CRISPRa) libraries, which use the ability of proteolytically deactivated Cas9-fusion proteins (dCas9) to bind target DNA, which means that a gene of interest is not cut but is over-expressed or repressed. It made CRISPR/Cas9 system even more interesting in gene editing. Inactive dCas9 protein modulate gene expression by targeting dCas9-repressors or activators toward promoter or transcriptional start sites of target genes. For repressing genes Cas9 can be fused to KRAB effector domain that makes complex with gRNA, whereas CRISPRa utilizes dCas9 fused to different transcriptional activation domains, which are further directed by gRNA to promoter regions to upregulate expression.^{[136][137][138]}

Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Cas9 can be easily introduced into the target cells along with sgRNA via plasmid transfection in order to model the spread of diseases and the cell's response to and defense against infection.^[139] The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function and mutation effects, all while avoiding the off-target mutations typically observed with older methods of genetic engineering.^{[citation needed]}

The CRISPR and Cas9 revolution in genomic modeling does not extend only to mammals. Traditional genomic models such as Drosophila melanogaster, one of the first model organisms, have seen further refinement in their resolution with the use of Cas9.^[139] Cas9 uses cell-specific promoters allowing a controlled use of the Cas9. Cas9 is an accurate method of treating diseases d