scholarly journals Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

2019 ◽  
Author(s):  
Zhuqing Wang ◽  
Yue Wang ◽  
Shawn Wang ◽  
Andrew J Gorzalski ◽  
Hayden McSwiggin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Editing Efficiency ◽  
Protospacer Adjacent Motif ◽  
Summary Statement ◽  
Strict Requirement ◽  
Moraxella Bovoculi ◽  
Target Effects

AbstractDespite many advantages over Cas9, Cas12a has not been widely used in genome editing in mammalian cells largely due to its strict requirement of the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM sequences and with minimal on-target mutations and close to 100% editing efficiency when crRNAs of 23nt spacers were used.Summary statementCRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and high overall knock-in efficiency.

scholarly journals Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects

2021 ◽  
Vol 12 ◽  
Author(s):  
Yufan Xu ◽  
Xiaorong Peng ◽  
Yanghao Zheng ◽  
Changzhong Jin ◽  
Xiangyun Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Host Cells ◽  
Preliminary Evaluation ◽  
Major Barrier ◽  
Editing Efficiency ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Target Effects

Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.

scholarly journals Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity

2020 ◽  
Vol 6 (6) ◽  
pp. eaay0187 ◽  
Author(s):  
Sabine Aschenbrenner ◽  
Stefan M. Kallenberger ◽  
Mareike D. Hoffmann ◽  
Adrian Huck ◽  
Roland Eils ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Transient Transfection ◽  
Target Specificity ◽  
Guide Rnas ◽  
Modeling And Experiments ◽  
Fine Tune ◽  
Target Effects

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

scholarly journals Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Soo-Ji Park ◽  
Tae Yeong Jeong ◽  
Seung Kyun Shin ◽  
Da Eun Yoon ◽  
Soo-Yeon Lim ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Genome Editing ◽  
Mouse Models ◽  
Targeted Mutagenesis ◽  
Mutant Mice ◽  
Germline Transmission ◽  
Dwarf Phenotype ◽  
Editing Efficiency ◽  
Mouse Cells ◽  
Target Effects

AbstractPrime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.

scholarly journals Direct Delivery of Cas9-sgRNA Ribonucleoproteins into Cells Using a Nanoneedle Array

2019 ◽  
Vol 9 (5) ◽  
pp. 965 ◽  
Author(s):  
Ayana Yamagishi ◽  
Daisuke Matsumoto ◽  
Yoshio Kato ◽  
Yuki Honda ◽  
Mone Morikawa ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Genome Editing ◽  
High Throughput ◽  
Mammalian Cells ◽  
Non Invasive ◽  
Nanoneedle Array ◽  
Novel Method ◽  
Direct Delivery ◽  
Target Effects ◽  
2D Array

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a powerful and widely used tool for genome editing. Recently, it was reported that direct delivery of Cas9-sgRNA ribonucleoproteins (RNPs) reduced off-target effects. Therefore, non-invasive, high-throughput methods are needed for direct delivery of RNPs into cells. Here, we report a novel method for direct delivery of RNPs into cells using a nanostructure with a high-aspect-ratio and uniform nanoneedles. This nanostructure is composed of tens of thousands of nanoneedles laid across a 2D array. Through insertion of the nanoneedle array previously adsorbed with Cas9-sgRNA, it was possible to deliver RNPs directly into mammalian cells for genome editing.

scholarly journals CRISPR Del/Rei: A simple, flexible and efficient pipeline for scarless genome editing

2021 ◽  
Author(s):  
Marah H. Wahbeh ◽  
Kyra L. Feuer ◽  
Sara Abdollahi ◽  
Christian Yovo ◽  
Eman Rabie ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Editing Efficiency ◽  
Manual Selection ◽  
Variant Alleles ◽  
Induced Pluripotent ◽  
The Individual ◽  
User Friendly ◽  
Selection Of ◽  
Target Effects

Scarless genome editing is an important tool for the accurate recapitulation of genetic variation in human disease models. Various CRISPR/Cas9-based scarless editing methods have been reported. However, some of these methods have low editing efficiency (1-5%) and require manual selection of hundreds of clones to reach the desired number. Other protocols use large selection cassettes with laborious vector assembly and specialized reagents and equipment, or have poorly understood off-target effects. To address these limitations, we developed a simple, highly efficient scarless editing strategy to edit DNA sequences in induced pluripotent stem cells, which we call CRISPR Del/Rei. This novel editing strategy consists of a two-step deletion-reinsertion strategy that produces isogenic clones in ~8 weeks using accessible, user-friendly reagents. The editing efficiency ranges from ~15–100% for Step 1 and ~5–20% for Step 2 after selection, which greatly reduces the amount of required manual clone isolation. Screening the transfected bulk cells and the individual clones is rapid and simple, consisting of PCR and gel electrophoresis. Despite the two editing steps, off-target effects are rare. Additionally, the experiment is well-controlled because the same protocol generates isogenic clones carrying all variant alleles. In this way, CRISPR Del/Rei serves as a valuable addition to the evolving CRISPR/Cas9 gene-editing toolset.

scholarly journals Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

2020 ◽  
Author(s):  
Wei Wang ◽  
Bin Tian ◽  
Qianli Pan ◽  
Yueying Chen ◽  
Fei He ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Genome Editing ◽  
Agronomic Traits ◽  
Wheat Genome ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Endogenous Genes

AbstractThe development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

scholarly journals Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jongjin Park ◽  
Jiyoung Yoon ◽  
Daekee Kwon ◽  
Mi-Jung Han ◽  
Sunmee Choi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fusion Proteins ◽  
Plant Cells ◽  
Targeted Mutagenesis ◽  
Chimeric Proteins ◽  
Editing Efficiency ◽  
Noticeable Increase ◽  
The Cost ◽  
Target Effects

AbstractEfforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5′-to-3′ exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.

scholarly journals Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases

2020 ◽  
Vol 48 (7) ◽  
pp. 3722-3733 ◽  
Author(s):  
Eszter Tóth ◽  
Éva Varga ◽  
Péter István Kulcsár ◽  
Virág Kocsis-Jutka ◽  
Sarah Laura Krausz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Plant Cells ◽  
Dependent Manner ◽  
Protospacer Adjacent Motif

Abstract The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

scholarly journals Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity

2019 ◽  
Vol 116 (42) ◽  
pp. 20969-20976 ◽  
Author(s):  
Yuanyan Tan ◽  
Athena H. Y. Chu ◽  
Siyu Bao ◽  
Duc Anh Hoang ◽  
Firaol Tamiru Kebede ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Editing ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Genome Wide ◽  
Protospacer Adjacent Motif ◽  
Targeted Deep Sequencing ◽  
Targeting Accuracy ◽  
Target Effects

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

scholarly journals CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

2019 ◽  
Author(s):  
Zhiliang Yang ◽  
Harley Edwards ◽  
Peng Xu
Keyword(s):  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Oleaginous Yeast ◽  
Standard Type ◽  
Editing Efficiency ◽  
Color Changes ◽  
Indel Mutation ◽  
Protospacer Adjacent Motif ◽  
Colony Color ◽  
First Time

AbstractCRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5’-phosphate decarboxylase (∼96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3’ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.

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