Prime editing

Last updated

Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. [1]

Contents

The technology has received mainstream press attention due to its potential uses in medical genetics. It utilizes methodologies similar to precursor genome editing technologies, including CRISPR/Cas9 and base editors. Prime editing has been used on some animal models of genetic disease [2] [3] [4] and plants. [5]

Genome editing

Components

Components of prime editing Components of prime editing.png
Components of prime editing

Prime editing involves three major components: [1]

Mechanism

Prime editing mechanism Prime editing mechanism.png
Prime editing mechanism

Genomic editing takes place by transfecting cells with the pegRNA and the fusion protein. Transfection is often accomplished by introducing vectors into a cell. Once internalized, the fusion protein nicks the target DNA sequence, exposing a 3’-hydroxyl group that can be used to initiate (prime) the reverse transcription of the RT template portion of the pegRNA. This results in a branched intermediate that contains two DNA flaps: a 3’ flap that contains the newly synthesized (edited) sequence, and a 5’ flap that contains the dispensable, unedited DNA sequence. The 5’ flap is then cleaved by structure-specific endonucleases or 5’ exonucleases. This process allows 3’ flap ligation, and creates a heteroduplex DNA composed of one edited strand and one unedited strand. The reannealed double stranded DNA contains nucleotide mismatches at the location where editing took place. In order to correct the mismatches, the cells exploit the intrinsic mismatch repair (MMR) mechanism, with two possible outcomes: (i) the information in the edited strand is copied into the complementary strand, permanently installing the edit; (ii) the original nucleotides are re-incorporated into the edited strand, excluding the edit. [1]

Development process

During the development of this technology, several modifications were done to the components, in order to increase its effectiveness. [1]

Prime editor 1

In the first system, a wild-type Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase was fused to the Cas9 H840A nickase C-terminus. Detectable editing efficiencies were observed. [1]

Prime editor 2

In order to enhance DNA-RNA affinity, enzyme processivity, and thermostability, five amino acid substitutions were incorporated into the M-MLV reverse transcriptase. The mutant M-MLV RT was then incorporated into PE1 to give rise to (Cas9 (H840A)-M-MLV RT(D200N/L603W/T330P/T306K/W313F)). Efficiency improvement was observed over PE1. [1]

PE3 system for prime editing PE3 system for prime editing.png
PE3 system for prime editing

Prime editor 3

Despite its increased efficacy, the edit inserted by PE2 might still be removed due to DNA mismatch repair of the edited strand. To avoid this problem during DNA heteroduplex resolution, an additional single guide RNA (sgRNA) is introduced. This sgRNA is designed to match the edited sequence introduced by the pegRNA, but not the original allele. It directs the Cas9 nickase portion of the fusion protein to nick the unedited strand at a nearby site, opposite to the original nick. Nicking the non-edited strand causes the cell's natural repair system to copy the information in the edited strand to the complementary strand, permanently installing the edit. [1] However, there are drawbacks to this system as nicking the unaltered strand can lead to additional undesired indels. [9]

Prime Editor 4

Prime editor 4 utilizes the same machinery as PE2, but also includes a plasmid that encodes for dominant negative MMR protein MLH1. Dominant negative MLH1 is able to essentially knock out endogenous MLH1 by inhibition, thereby reducing cellular MMR response and increasing prime editing efficiency. [9]

Prime Editor 5

Prime editor 5 utilizes the same machinery as PE3, but also includes a plasmid that encodes for dominant negative MLH1. Like PE4, this allows for a knockdown of endogenous MMR response, increasing the efficiency of prime editing. [9]

Nuclease Prime Editor

Nuclease Prime Editor uses Cas9 nuclease instead of Cas9(H840A) nickase. Unlike prime editor 3 (PE3) that requires dual-nick at both DNA strands to induce efficient prime editing, Nuclease Prime Editor requires only a single pegRNA since the single-gRNA already creates double-strand break instead of single-strand nick. [10]

Twin Prime Editing

The "twin prime editing" (twinPE) mechanism reported in 2021 allows editing large sequences of DNA – sequences as large as genes – which addresses the method's key drawback. It uses a prime editor protein and two prime editing guide RNAs. [11] [12] [ more detail needed ]

History

Prime editing was developed in the lab of David R. Liu at the Broad Institute and disclosed in Anzalone et al. (2019). [13] Since then prime editing and the research that produced it have received widespread scientific acclaim, [14] [6] [15] being called "revolutionary" [7] and an important part of the future of editing. [13]

Development of epegRNAs

Prime editing efficiency can be increased with the use of engineered pegRNAs (epegRNAs). One common issue with traditional pegRNAs is degradation of the 3' end, leading to decreased PE efficiency. epegRNAs have a structured RNA motif added to their 3' end to prevent degradation. [16]

Implications

Although additional research is required to improve the efficiency of prime editing, the technology offers promising scientific improvements over other gene editing tools. The prime editing technology has the potential to correct the vast majority of pathogenic alleles that cause genetic diseases, as it can repair insertions, deletions, and nucleotide substitutions. [1]

Advantages

The prime editing tool offers advantages over traditional gene editing technologies. CRISPR/Cas9 edits rely on non-homologous end joining (NHEJ) or homology-directed repair (HDR) to fix DNA breaks, while the prime editing system employs DNA mismatch repair. This is an important feature of this technology given that DNA repair mechanisms such as NHEJ and HDR, generate unwanted, random insertions or deletions (INDELs). These are byproducts that complicate the retrieval of cells carrying the correct edit. [1] [17]

The prime system introduces single-stranded DNA breaks instead of the double-stranded DNA breaks observed in other editing tools, such as base editors. Collectively, base editing and prime editing offer complementary strengths and weaknesses for making targeted transition mutations. Base editors offer higher editing efficiency and fewer INDEL byproducts if the desired edit is a transition point mutation and a PAM sequence exists roughly 15 bases from the target site. However, because the prime editing technology does not require a precisely positioned PAM sequence to target a nucleotide sequence, it offers more flexibility and editing precision. Remarkably, prime editors allow all types of substitutions, transitions and transversions to be inserted into the target sequence. [1] [17] Cytosine base editing and adenine BE can already perform precise base transitions but for base transversions there have been no good options. Prime editing performs transversions with good usability. PE can insert up to 44bp, delete up to 80, or combinations thereof. [7]

Because the prime system involves three separate DNA binding events (between (i) the guide sequence and the target DNA, (ii) the primer binding site and the target DNA, and (iii) the 3’ end of the nicked DNA strand and the pegRNA), it has been suggested to have fewer undesirable off-target effects than CRISPR/Cas9. [1] [17]

Limitations

There is considerable interest in applying gene-editing methods to the treatment of diseases with a genetic component. However, there are multiple challenges associated with this approach. An effective treatment would require editing of a large number of target cells, which in turn would require an effective method of delivery and a great level of tissue specificity. [1] [18]

As of 2019, prime editing looks promising for relatively small genetic alterations, but more research needs to be conducted to evaluate whether the technology is efficient in making larger alterations, such as targeted insertions and deletions. Larger genetic alterations would require a longer RT template, which could hinder the efficient delivery of pegRNA to target cells. Furthermore, a pegRNA containing a long RT template could become vulnerable to damage caused by cellular enzymes. [1] [18] Prime editing in plants suffers from low efficiency ranging from zero to a few percent and needs significant improvement. [19]

Some of these limitations have been mitigated by recent improvements to the prime editors, [2] [20] including motifs that protect pegRNAs from degradation. [21] Further research is needed before prime editing could be used to correct pathogenic alleles in humans. [1] [18] Research has also shown that inhibition of certain MMR proteins, including MLH1 can improve prime editing efficiency. [9]

Delivery method

Base editors used for prime editing require delivery of both a protein and RNA molecule into living cells. Introducing exogenous gene editing technologies into living organisms is a significant challenge. One potential way to introduce a base editor into animals and plants is to package the base editor into a viral capsid. The target organism can then be transduced by the virus to synthesize the base editor in vivo. Common laboratory vectors of transduction such as lentivirus cause immune responses in humans, so proposed human therapies often centered around adeno-associated virus (AAV) because AAV infections are largely asymptomatic. Unfortunately, the effective packaging capacity of AAV vectors is small, approximately 4.4kb not including inverted terminal repeats. [22] As a comparison, an SpCas9-reverse transcriptase fusion protein is 6.3kb, [1] [23] which does not even account for the lengthened guide RNA necessary for targeting and priming the site of interest. However, successful delivery in mice has been achieved by splitting the editor into two AAV vectors [2] [3] [4] [24] or by using an adenovirus, [3] which has a larger packaging capacity.

Applications

Prime editors may be used in gene drives. A prime editor may be incorporated into the Cleaver half of a Cleave and Rescue /ClvR system. In this case it is not meant to perform a precise alteration but instead to merely disrupt. [25]

PE is among recently introduced technologies which allow the transfer of single-nucleotide polymorphisms (SNPs) from one individual crop plant to another. PE is precise enough to be used to recreate an arbitrary SNP in an arbitrary target, [14] including deletions, insertions, and all 12 point mutations without also needing to perform a double-stranded break or carry a donating template. [6]

See also

Related Research Articles

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

<span class="mw-page-title-main">Insertion (genetics)</span> Type of mutation

In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.

<span class="mw-page-title-main">CRISPR</span> Family of DNA sequence found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, gRNAs are a part of the CRISPR-Cas system that serves as an adaptive immune defense that protects the organism from viruses. Here the short gRNAs serve as detectors of foreign DNA and direct the Cas-enzymes that degrades the foreign nucleic acid.

Missense mRNA is a messenger RNA bearing one or more mutated codons that yield polypeptides with an amino acid sequence different from the wild-type or naturally occurring polypeptide. Missense mRNA molecules are created when template DNA strands or the mRNA strands themselves undergo a missense mutation in which a protein coding sequence is mutated and an altered amino acid sequence is coded for.

<span class="mw-page-title-main">Insert (molecular biology)</span>

In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">CRISPR interference</span> Genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

<span class="mw-page-title-main">Epigenome editing</span>

Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

<span class="mw-page-title-main">Cas12a</span> DNA-editing technology

Cas12a is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of viruses and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases use an RNA to target nucleic acid in a specific and programmable matter. In the organisms from which it originates, this guide RNA is a copy of a piece of foreign nucleic acid that previously infected the cell.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs).

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing standing 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 and/or new ones added in vivo.

<span class="mw-page-title-main">Genome-wide CRISPR-Cas9 knockout screens</span> Research tool in genomics

Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.

The Fanzor (Fz) protein is an eukaryotic, RNA-guided DNA endonuclease, which means it is a type of DNA cutting enzyme that uses RNA to target genes of interest. It has been recently discovered and explored in a number of studies. In bacteria, RNA-guided DNA endonuclease systems, such as the CRISPR/Cas system, serve as an immune system to prevent infection by cutting viral genetic material. Currently, CRISPR/Cas9-mediated's DNA cleavage has extensive application in biological research, and wide-reaching medical potential in human gene editing.

References

  1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R. (21 October 2019). "Search-and-replace genome editing without double-strand breaks or donor DNA". Nature. 576 (7785): 149–157. Bibcode:2019Natur.576..149A. doi:10.1038/s41586-019-1711-4. PMC   6907074 . PMID   31634902.
  2. 1 2 3 Liu, Pengpeng; Liang, Shun-Qing; Zheng, Chunwei; Mintzer, Esther; Zhao, Yan G.; Ponnienselvan, Karthikeyan; Mir, Aamir; Sontheimer, Erik J.; Gao, Guangping; Flotte, Terence R.; Wolfe, Scot A. (2021-04-09). "Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice". Nature Communications. 12 (1): 2121. Bibcode:2021NatCo..12.2121L. doi:10.1038/s41467-021-22295-w. ISSN   2041-1723. PMC   8035190 . PMID   33837189.
  3. 1 2 3 Böck, Desirée; Rothgangl, Tanja; Villiger, Lukas; Schmidheini, Lukas; Mathis, Nicholas; Ioannidi, Eleonora; Kreutzer, Susanne; Kontarakis, Zacharias; Rimann, Nicole; Grisch-Chan, Hiu Man; Thöny, Beat (2021-08-17). "Treatment of a metabolic liver disease by in vivo prime editing in mice": 2021.08.17.456632. doi:10.1101/2021.08.17.456632. S2CID   237218057.{{cite journal}}: Cite journal requires |journal= (help)
  4. 1 2 Jang, Hyewon; Jo, Dong Hyun; Cho, Chang Sik; Shin, Jeong Hong; Seo, Jung Hwa; Yu, Goosang; Gopalappa, Ramu; Kim, Daesik; Cho, Sung-Rae; Kim, Jeong Hun; Kim, Hyongbum Henry (2021-08-26). "Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases". Nature Biomedical Engineering. 6 (2): 181–194. doi:10.1038/s41551-021-00788-9. ISSN   2157-846X. PMID   34446856. S2CID   237321703.
  5. ↑ Biswas, Sudip; Bridgeland, Aya; Irum, Samra; Thomson, Michael J.; Septiningsih, Endang M. (29 August 2022). "Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity". International Journal of Molecular Sciences. 23 (17): 9809.
  6. 1 2 3 Lin, Xueqiu; Chemparathy, Augustine; La Russa, Marie; Daley, Timothy; Qi, Lei S. (2020-07-20). "Computational Methods for Analysis of Large-Scale CRISPR Screens". Annual Review of Biomedical Data Science . 3 (1). Annual Reviews: 137–162. doi: 10.1146/annurev-biodatasci-020520-113523 . ISSN   2574-3414. S2CID   225570135.
  7. 1 2 3 Zhu, Haocheng; Li, Chao; Gao, Caixia (2020-09-24). "Applications of CRISPR–Cas in agriculture and plant biotechnology". Nature Reviews Molecular Cell Biology . 21 (11). Nature Portfolio: 661–677. doi:10.1038/s41580-020-00288-9. ISSN   1471-0072. PMID   32973356. S2CID   221918795.
  8. ↑ Ran, F. Ann; Hsu, Patrick D.; Lin, Chie-Yu; Gootenberg, Jonathan S.; Konermann, Silvana; Trevino, Alexandro E.; Scott, David A.; Inoue, Azusa; Matoba, Shogo; Zhang, Yi; Zhang, Feng (September 2013). "Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity". Cell. 154 (6): 1380–1389. doi:10.1016/j.cell.2013.08.021. PMC   3856256 . PMID   23992846.
  9. 1 2 3 4 Chen, Peter (October 28, 2021). "Enhanced Prime Editing Systems By Manipulating Cellular Determinants of Editing Outcomes". Cell. 184 (22): 5635–5652.e29. doi:10.1016/j.cell.2021.09.018. PMC   8584034 . PMID   34653350.
  10. ↑ Adikusuma, Fatwa; Lushington, Caleb; Arudkumar, Jayshen; Godahewa, Gelshan; Chey, Yu C J; Gierus, Luke; Geiger, Ashleigh; Jain, Yatish; Reti, Daniel; Wilson, Laurence O W; Bower, Denis C; Thomas, Paul Q (17 September 2021). "Optimized nickase- and nuclease-based prime editing in human and mouse cells". Nucleic Acids Research. 49 (18): 10785–10795. doi:10.1093/nar/gkab792. PMC   8501948 . PMID   34534334.
  11. ↑ Dicorato, Allessandra. "New prime editing system inserts entire genes in human cells". Broad Institute of MIT. Retrieved 16 January 2022.
  12. ↑ Anzalone, Andrew V.; Gao, Xin D.; Podracky, Christopher J.; Nelson, Andrew T.; Koblan, Luke W.; Raguram, Aditya; Levy, Jonathan M.; Mercer, Jaron A. M.; Liu, David R. (9 December 2021). "Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing". Nature Biotechnology. 40 (5): 731–740. doi:10.1038/s41587-021-01133-w. ISSN   1546-1696. PMC   9117393 . PMID   34887556. S2CID   245012407.
  13. 1 2 Graff, Gregory D.; Sherkow, Jacob S. (2020-08-31). "Models of Technology Transfer for Genome-Editing Technologies". Annual Review of Genomics and Human Genetics . 21 (1). Annual Reviews: 509–534. doi:10.1146/annurev-genom-121119-100145. hdl: 2142/110346 . ISSN   1527-8204. PMID   32151165. S2CID   212652569.
  14. 1 2 Soyk, Sebastian; Benoit, Matthias; Lippman, Zachary B. (2020-11-23). "New Horizons for Dissecting Epistasis in Crop Quantitative Trait Variation". Annual Review of Genetics . 54 (1). Annual Reviews: 287–307. doi:10.1146/annurev-genet-050720-122916. ISSN   0066-4197. PMID   32870731. S2CID   221467135.
  15. ↑ Faculty Opinions of Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R. (2019). "Search-and-replace genome editing without double-strand breaks or donor DNA". Nature. 576 (7785): 149–157. Bibcode:2019Natur.576..149A. doi:10.1038/s41586-019-1711-4. PMC   6907074 . PMID   31634902 . Retrieved 2021-11-13.
  16. ↑ Nelson, James (October 4, 2021). "Engineered pegRNAs improve prime editing efficiency". Nature Biotechnology. 40 (432): 402–410. doi:10.1038/s41587-021-01039-7. PMC   8930418 . PMID   34608327.
  17. 1 2 3 Sheridan, Cormac (7 November 2019). "Gene editing enters 'prime' time". Nature Biotechnology. doi:10.1038/d41587-019-00032-5. PMID   33154577. S2CID   209564966.
  18. 1 2 3 "Scientist David Liu takes your questions on CRISPR and prime editing". STAT. 2019-11-06. Retrieved 2020-02-28.
  19. ↑ Molla, Kutubuddin; Sretenovic, Simon; Bansal, Kailash C.; Qi, Yiping (13 September 2021). "Precise plant genome editing using base editors and prime editors". Nature Plants. 7 (9): 1166–1187. doi:10.1038/s41477-021-00991-1. PMID   34518669. S2CID   237503774.
  20. ↑ Chen, Peter J.; Hussmann, Jeffrey A.; Yan, Jun; Knipping, Friederike; Ravisankar, Purnima; Chen, Pin-Fang; Chen, Cidi; Nelson, James W.; Newby, Gregory A.; Sahin, Mustafa; Osborn, Mark J. (October 2021). "Enhanced prime editing systems by manipulating cellular determinants of editing outcomes". Cell. 184 (22): 5635–5652.e29. doi:10.1016/j.cell.2021.09.018. ISSN   0092-8674. PMC   8584034 . PMID   34653350.
  21. ↑ Nelson, James W.; Randolph, Peyton B.; Shen, Simon P.; Everette, Kelcee A.; Chen, Peter J.; Anzalone, Andrew V.; An, Meirui; Newby, Gregory A.; Chen, Jonathan C.; Hsu, Alvin; Liu, David R. (2021-10-04). "Engineered pegRNAs improve prime editing efficiency". Nature Biotechnology. 40 (3): 402–410. doi:10.1038/s41587-021-01039-7. ISSN   1546-1696. PMC   8930418 . PMID   34608327. S2CID   238356160.
  22. ↑ Wu, Zhijian; Yang, Hongyan; Colosi, Peter (2010). "Effect of Genome Size on AAV Vector Packaging". Molecular Therapy. 18 (1): 80–86. doi:10.1038/mt.2009.255. PMC   2839202 . PMID   19904234.
  23. ↑ "Addgene: PCMV-PE2".
  24. ↑ Zhi, Shengyao; Chen, Yuxi; Wu, Guanglan; Wen, Jinkun; Wu, Jinni; Liu, Qianyi; Li, Yang; Kang, Rui; Hu, Sihui; Wang, Jiahui; Liang, Puping (July 2021). "Dual-AAV delivering split prime editor system for in vivo genome editing". Molecular Therapy. 30 (1): 283–294. doi:10.1016/j.ymthe.2021.07.011. ISSN   1525-0016. PMC   8753371 . PMID   34298129. S2CID   236212353.
  25. ↑ Hay, Bruce A.; Oberhofer, Georg; Guo, Ming (2021-01-07). "Engineering the Composition and Fate of Wild Populations with Gene Drive". Annual Review of Entomology . 66 (1). Annual Reviews: 407–434. doi: 10.1146/annurev-ento-020117-043154 . ISSN   0066-4170. PMID   33035437. S2CID   222257628.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.