Prime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA

Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)–based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.

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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

10.20944/preprints202007.0578.v1 ◽

2020 ◽

Author(s):

Anindya Bandyopadhyay ◽

Nagesh Kancharla ◽

vivek javalkote ◽

santanu dasgupta ◽

Thomas Brutnell

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Temperature Stability ◽

Double Strand Breaks ◽

Plant Genome ◽

Strand Breaks ◽

Guide Rna ◽

Versatile Tool ◽

Type V ◽

Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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CRISPR base editors: genome editing without double-stranded breaks

Biochemical Journal ◽

10.1042/bcj20170793 ◽

2018 ◽

Vol 475 (11) ◽

pp. 1955-1964 ◽

Cited By ~ 79

Author(s):

Ayman Eid ◽

Sahar Alshareef ◽

Magdy M. Mahfouz

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genetic Diseases ◽

Great Promise ◽

Cytidine Deaminases ◽

Strand Breaks ◽

Base Editing ◽

Potential Applications ◽

Short Palindromic Repeat ◽

Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

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TALEN-mediated genome editing: prospects and perspectives

Biochemical Journal ◽

10.1042/bj20140295 ◽

2014 ◽

Vol 462 (1) ◽

pp. 15-24 ◽

Cited By ~ 65

Author(s):

David A. Wright ◽

Ting Li ◽

Bing Yang ◽

Martin H. Spalding

Keyword(s):

Genome Editing ◽

Double Strand Breaks ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Strand Breaks ◽

Natural Processes ◽

Current State ◽

A Genome ◽

Dna Dsbs ◽

Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

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Faculty Opinions recommendation of Search-and-replace genome editing without double-strand breaks or donor DNA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736778482.793579890 ◽

2020 ◽

Author(s):

Yousin Suh

Keyword(s):

Genome Editing ◽

Double Strand Breaks ◽

Strand Breaks

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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

10.1101/161109 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alexandre Paix ◽

Andrew Folkmann ◽

Daniel H Goldman ◽

Heather Kulaga ◽

Michael Grzelak ◽

...

Keyword(s):

Genome Editing ◽

Rational Design ◽

High Efficiency ◽

Fluorescent Protein ◽

Double Strand Breaks ◽

Template Switching ◽

Dna Breaks ◽

Simple Method ◽

Strand Breaks ◽

Strand Annealing

AbstractThe RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.SignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with ∼35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.

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Application of the Modified Cytosine Base-Editing in the Cultured Cells of Bama Minipig

10.21203/rs.3.rs-473930/v1 ◽

2021 ◽

Author(s):

Jia-sheng Pan ◽

Zi-sheng Lin ◽

Jian-cong Wen ◽

Jian-feng Guo ◽

Xia-hui Wu ◽

...

Keyword(s):

Genome Editing ◽

Base Pair ◽

Double Strand Breaks ◽

Fibroblast Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Single Base ◽

Lean Meat ◽

Base Editing ◽

Target Effects

Abstract Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

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Simultaneous precise editing of multiple genes in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz669 ◽

2019 ◽

Vol 47 (19) ◽

pp. e116-e116 ◽

Cited By ~ 17

Author(s):

Stephan Riesenberg ◽

Manjusha Chintalapati ◽

Dominik Macak ◽

Philipp Kanis ◽

Tomislav Maricic ◽

...

Keyword(s):

Genome Editing ◽

Kinase Inhibitor ◽

Double Strand Breaks ◽

End Joining ◽

Nucleotide Substitutions ◽

Strand Breaks ◽

Homology Directed Repair ◽

A Genome ◽

Dependent Protein ◽

Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

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Microhomology-mediated CRISPR/Cas9-based method for genome editing in fission yeast

10.1101/498402 ◽

2018 ◽

Author(s):

Aki Hayashi ◽

Katsunori Tanaka

Keyword(s):

Fission Yeast ◽

Genome Editing ◽

High Frequency ◽

Target Genes ◽

Point Mutations ◽

Inducible Promoter ◽

Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Guide Rna

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the microhomology mediated end joining (MMEJ)-based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the MMEJ-mediated genome editing and found that the MMEJ-mediated method provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing microhomology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

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High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

10.1101/2020.05.12.091991 ◽

2020 ◽

Author(s):

Karthik Murugan ◽

Arun S. Seetharam ◽

Andrew J. Severin ◽

Dipali G. Sashital

Keyword(s):

Genome Editing ◽

High Throughput ◽

Double Strand Breaks ◽

Target Sequence ◽

Wild Type ◽

Strand Breaks ◽

Cleavage Assay ◽

Target Sequences

AbstractCas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

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