scholarly journals Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

2021 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Chan Hyoung Kim ◽  
Yeounsun Oh ◽  
Lee Wha Gwon ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Deoxyribonucleic Acid ◽  
Specific Gene ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Wild Type ◽  
Dna Cleaving ◽  
Target Dna ◽  
Near Future

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

2021 ◽  
Vol 7 (7) ◽  
pp. 505
Author(s):  
Ping Zhang ◽  
Yu Wang ◽  
Chenxi Li ◽  
Xiaoyu Ma ◽  
Lan Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fungal Pathogens ◽  
Gene Editing ◽  
Recombination Frequency ◽  
Target Sequence ◽  
Widespread Application ◽  
Genetic Studies ◽  
Efficient Gene ◽  
Cryptococcus Species ◽  
Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

scholarly journals Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

Haematologica ◽  
2021 ◽  
Author(s):  
Dan Lu ◽  
Xiuli Gong ◽  
Yudan Fang ◽  
Xinbing Guo ◽  
Yanwen Chen ◽  
...  
Keyword(s):  
Survival Rate ◽  
Genome Editing ◽  
Molecular Mechanism ◽  
Splice Site ◽  
Rna Splicing ◽  
Gene Editing ◽  
Clinical Symptoms ◽  
Rna Expression ◽  
Wild Type ◽  
Acceptor Splice Site

β654-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of total β-thalassemia cases in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nt by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNAs and Cas9 mRNAs into 1-cell embryos of β654 or control mice. 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited-β654 founders and their offspring, were significantly improved compared to the non-edited β654 mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and live-born homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide a groundwork for the exploration of β654-thalassemia therapy in the future.

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

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

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.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Katarzyna M. Soczek ◽  
Gavin J. Knott ◽  
Eva Nogales ◽  
Jennifer A. Doudna
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

scholarly journals Gene Replacement Therapies for Genodermatoses: A Status Quo

2021 ◽  
Vol 12 ◽  
Author(s):  
Ulrich Koller ◽  
Johann W. Bauer
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Gene Editing ◽  
Gene Replacement ◽  
Mechanical Trauma ◽  
Wild Type ◽  
Number Of Patients ◽  
Genes Encoding ◽  
Skin Fragility ◽  
Recent Developments

Epidermolysis bullosa (EB) is a genodermatosis, characterized by the formation of extended blisters and lesions on the skin and mucous membranes upon minimal mechanical trauma. The disease is caused by mutations in genes encoding proteins that are essential for skin stability. Functional impairment, reduction, or absence of one of these proteins results in skin fragility due to reduced connectivity between dermis and epidermis. Currently, gene therapy represents the only treatment option with the potential to cure this severe blistering skin disease. Two promising forms of gene therapy are potentially feasible for EB: gene replacement and genome editing. While genome editing for genodermatoses remains at the preclinical stage, gene replacement approaches are clinically advanced and have been applied already to a small number of patients with junctional and dystrophic forms of EB. Here, the viral transduction of the “wild-type” transgene into skin stem cells, followed by autologous grafting of corrected epidermal sheets, led to the regeneration of stable skin. Recent developments regarding designer nuclease-based gene editing strategies enable the establishment of alternative options to restore the gene function in genodermatoses. This is particularly true in cases wherein genetic constellation hinders gene therapy-based gene replacement.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Jennifer Doudna ◽  
Joshua Cofsky ◽  
Katarzyna Soczek ◽  
Gavin Knott ◽  
Eva Nogales
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

Abstract In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

scholarly journals CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

2021 ◽  
Author(s):  
Varsha Kumari ◽  
Priyanka Kumawat ◽  
Sharanabasappa Yeri ◽  
Shyam Singh Rajput
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Crop Improvement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Ligase Iv ◽  
A Genome ◽  
Target Dna ◽  
Non Homologous End Joining ◽  
Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

scholarly journals In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Woo-Chan Ahn ◽  
Kwang-Hyun Park ◽  
In Seon Bak ◽  
Hyung-Nam Song ◽  
Yan An ◽  
...  
Keyword(s):  
Genome Editing ◽  
Knockout Mice ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Essential Gene ◽  
Bacterial Species ◽  
A Genome ◽  
Target Dna ◽  
Metal Cofactor

Abstract Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5′ target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.

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