A G-quadruplex motif at the 3′ end of sgRNAs improves CRISPR–Cas9 based genome editing efficiency

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

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Genome editing's potential target diseases in the cardiovascular field

10.31219/osf.io/gc23p ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Cardiovascular Diseases ◽

Genome Editing ◽

Target Cells ◽

Clinical Use ◽

Editing Efficiency ◽

Potential Target ◽

Genome Wide ◽

Significant Barrier

Two types of cardiovascular diseases can be cured or prevented using genome editing. The liver is the organ that has received the most attention in terms of clinical genome editing for cardiovascular diseases. Off-target mutagenesis is a concern of any form of genome editing. Off-target mutations in target cells or tissues may lead to undesirable functional phenotypes, including cancer. For therapeutic editing of the heart, the authors claim it's critical to achieve high editing efficiency at a chosen genomic site in a desired tissue. Off-target editing can be tested genome-wide unbiasedly using newer cell-based methods. For low off-target impact, well-designed gRNAs are important. The delivery of genome editors to target tissues and cells is a significant barrier to clinical use.

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A qPCR method for genome editing efficiency determination and single-cell clone screening in human cells

Scientific Reports ◽

10.1038/s41598-019-55463-6 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Bo Li ◽

Naixia Ren ◽

Lele Yang ◽

Junhao Liu ◽

Qilai Huang

Keyword(s):

Single Cell ◽

Genome Editing ◽

Cell Clone ◽

Editing Efficiency ◽

Base Editing ◽

Single Cell Clone ◽

Cell Clones ◽

Nucleotide Mismatch

AbstractCRISPR/Cas9 technology has been widely used for targeted genome modification both in vivo and in vitro. However, an effective method for evaluating genome editing efficiency and screening single-cell clones for desired modification is still lacking. Here, we developed this real time PCR method based on the sensitivity of Taq DNA polymerase to nucleotide mismatch at primer 3′ end during initiating DNA replication. Applications to CRISPR gRNAs targeting EMX1, DYRK1A and HOXB13 genes in Lenti-X 293 T cells exhibited comprehensive advantages. Just in one-round qPCR analysis using genomic DNA from cells underwent CRISPR/Cas9 or BE4 treatments, the genome editing efficiency could be determined accurately and quickly, for indel, HDR as well as base editing. When applied to single-cell clone screening, the genotype of each cell colony could also be determined accurately. This method defined a rigorous and practical way in quantify genome editing events.

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CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Agronomy ◽

10.3390/agronomy10071033 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1033 ◽

Cited By ~ 3

Author(s):

Jake Adolf V. Montecillo ◽

Luan Luong Chu ◽

Hanhong Bae

Keyword(s):

Genetic Engineering ◽

Genome Editing ◽

Gene Editing ◽

Fine Tuning ◽

Plant Genome ◽

Detection Methods ◽

Editing Efficiency ◽

Targeted Gene Editing ◽

Transgene Detection ◽

Selection Of

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

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Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

Genome Biology ◽

10.1186/s13059-020-02170-5 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Yuan-Yuan Jiang ◽

Yi-Ping Chai ◽

Min-Hui Lu ◽

Xiu-Li Han ◽

Qiupeng Lin ◽

...

Keyword(s):

Genome Editing ◽

Transgenic Rice ◽

Editing Efficiency ◽

Low Efficiency

Abstract Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

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Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes

Theriogenology ◽

10.1016/j.theriogenology.2017.11.030 ◽

2018 ◽

Vol 108 ◽

pp. 29-38 ◽

Cited By ~ 13

Author(s):

Masahiro Sato ◽

Maeda Kosuke ◽

Miyu Koriyama ◽

Emi Inada ◽

Issei Saitoh ◽

...

Keyword(s):

Genome Editing ◽

Editing Efficiency

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Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

PLoS ONE ◽

10.1371/journal.pone.0190192 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0190192 ◽

Cited By ~ 3

Author(s):

Michail Zaboikin ◽

Carl Freter ◽

Narasimhachar Srinivasakumar

Keyword(s):

High Resolution ◽

Genome Editing ◽

High Resolution Melt ◽

Editing Efficiency ◽

Gaussian Decomposition

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Engineering CRISPR–Cpf1 crRNAs and mRNAs to maximize genome editing efficiency

Nature Biomedical Engineering ◽

10.1038/s41551-017-0066 ◽

2017 ◽

Vol 1 (5) ◽

Cited By ~ 41

Author(s):

Bin Li ◽

Weiyu Zhao ◽

Xiao Luo ◽

Xinfu Zhang ◽

Chenglong Li ◽

...

Keyword(s):

Genome Editing ◽

Editing Efficiency

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Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

10.1101/176719 ◽

2017 ◽

Author(s):

Michail Zaboikin ◽

Carl Freter ◽

Narasimhachar Srinivasakumar

Keyword(s):

High Resolution ◽

Genome Editing ◽

Next Generation Sequencing Data ◽

Gaussian Component ◽

Sequencing Data ◽

High Resolution Melt ◽

Editing Efficiency ◽

Target Sites ◽

Corrected Data ◽

Derivatives Of

AbstractWe describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.629153 ◽

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.

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