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

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin 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 1,000 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. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

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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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Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

eLife ◽

10.7554/elife.04766 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 554

Author(s):

Steven Lin ◽

Brett T Staahl ◽

Ravi K Alla ◽

Jennifer A Doudna

Keyword(s):

Genome Engineering ◽

High Efficiency ◽

Embryonic Stem ◽

Human Cells ◽

Dna Breaks ◽

Site Specific ◽

Guide Rna ◽

Homology Directed Repair ◽

Double Strand Dna Breaks ◽

Cell Mortality

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

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Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

10.1101/2021.09.14.460298 ◽

2021 ◽

Author(s):

Jeffrey C Medley ◽

Shilpa Hebbar ◽

Joel T Sydzyik ◽

Anna Y. Zinovyeva

Keyword(s):

Caenorhabditis Elegans ◽

Genome Editing ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Paternal Genome ◽

Single Nucleotide ◽

C Elegans ◽

Homology Directed Repair ◽

Maternal Genome ◽

Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

10.1101/352260 ◽

2018 ◽

Author(s):

Gregoriy A. Dokshin ◽

Krishna S. Ghanta ◽

Katherine M. Piscopo ◽

Craig C. Mello

Keyword(s):

Green Fluorescent Protein ◽

Cost Effectiveness ◽

Genome Editing ◽

High Efficiency ◽

Fluorescent Protein ◽

C Elegans ◽

Multiple Loci ◽

Green Fluorescent ◽

Fluorescent Protein Tags ◽

Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

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The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

10.1101/628107 ◽

2019 ◽

Author(s):

Florian Veillet ◽

Laura Chauvin ◽

Marie-Paule Kermarrec ◽

François Sevestre ◽

Mathilde Merrer ◽

...

Keyword(s):

Solanum Tuberosum ◽

Genome Editing ◽

Genome Engineering ◽

Basic Research ◽

Loss Of Function ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Discrete Variation ◽

Tetraploid Potato ◽

Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

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Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

10.21203/rs.2.23248/v1 ◽

2020 ◽

Author(s):

Haseena Khan ◽

Megan C McDonald ◽

Simon J Willams ◽

Peter Solomon

Keyword(s):

Homologous Recombination ◽

Genome Editing ◽

Pathogenic Fungus ◽

Point Mutations ◽

Dna Breaks ◽

Gene Manipulation ◽

Homology Directed Repair ◽

Rnp Complex ◽

Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

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Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

10.1101/704197 ◽

2019 ◽

Author(s):

Geoffrey L. Rogers ◽

Hsu-Yu Chen ◽

Heidy Morales ◽

Paula M. Cannon

Keyword(s):

Homologous Recombination ◽

Progenitor Cells ◽

Genome Editing ◽

High Efficiency ◽

Zinc Finger Nucleases ◽

Dna Breaks ◽

Adeno Associated Virus ◽

Hematopoietic Stem ◽

Double Stranded Dna ◽

Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

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Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Open Biology ◽

10.1098/rsob.210241 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Yile Hao ◽

Qinhua Wang ◽

Jie Li ◽

Shihui Yang ◽

Yanli Zheng ◽

...

Keyword(s):

Genome Editing ◽

Large Scale ◽

Genome Engineering ◽

High Efficiency ◽

Helicase Domain ◽

Type I ◽

Strand Breaks ◽

Gene Insertion ◽

Type V

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

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Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

10.1101/2021.06.30.450566 ◽

2021 ◽

Author(s):

Manning Y. Huang ◽

Meenakshi B. Joshi ◽

Michael J Boucher ◽

Sujin Lee ◽

Liza C. Loza ◽

...

Keyword(s):

Homologous Recombination ◽

Cryptococcus Neoformans ◽

Genome Engineering ◽

Fluorescent Protein ◽

Pcr Amplification ◽

Sexual Cycle ◽

Calmodulin Binding ◽

Homology Directed Repair ◽

Recombination Efficiency ◽

Localization Signal

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or 'CnoCAS9') reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

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Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802343115 ◽

2018 ◽

Vol 115 (31) ◽

pp. E7379-E7388 ◽

Cited By ~ 21

Author(s):

Laura J. Smith ◽

Jason Wright ◽

Gabriella Clark ◽

Taihra Ul-Hasan ◽

Xiangyang Jin ◽

...

Keyword(s):

Stem Cell ◽

Homologous Recombination ◽

Genome Editing ◽

High Efficiency ◽

Dna Breaks ◽

Hematopoietic Stem ◽

A Genome ◽

Component Approach ◽

Il2rg Gene

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

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