689. Defining Genome-Wide Off-Target Cleavage Profiles of CRISPR-Cas RNA-Guided Nucleases Using GUIDE-Seq

Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nancy; Liebers, Matthew; Topkar, V.V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd

doi:10.1016/s1525-0016(16)34298-8

Cited by 3 publications

(4 citation statements)

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“…In cases where the mismatched off-target has mismatches only in the PAM-distal region, off-targets containing mismatches at position 1 are likely to be prevalent. This information is consistent with a recent publication (31) and further supports the need to consider position 1 in crRNA design rules. More target/off-target sets will need to be tested to predict precise bioinformatic rules for off-targeting, however the rapid ability to test many synthetic crRNAs per gene target and some understanding of mismatch tolerance and are essential to triage designs for highly specific and functional crRNAs.…”

Section: Discussionsupporting

confidence: 91%

“…Considering that either on-target or off-target activity will strictly require a PAM, (thus limiting the vast number of possible 1-, 2-, and 3-base-mismatched off-targets to only the subset which occur in the genome adjacent to a PAM) and that only a small fraction of these possible sites, in context of the current study, show activity, our results support the conclusion that off-targeting is not as much of an impediment to the use of CRISPR-Cas9 as was initially suggested. Several methods have recently emerged that enable profiling of genome-wide offtarget cleavage by CRISPR-Cas9 (31)(32)(33). While GUIDE-Seq may provide an approach to detect genome-wide off-target cleavage for single sgRNAs in a low throughput manner, our method allows detection of functional genome editing and a proxy method for off-targeting through numerous, unbiased mismatches to a crRNA with similar powerful sensitivity to these published methods but also in a high-throughput manner with respect to both targets and off-targets detected.…”

Section: Discussionmentioning

confidence: 99%

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Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity

Anderson¹,

Haupt²,

Schiel³

et al. 2015

Journal of Biotechnology

146

105

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The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity

Anderson¹,

Haupt²,

Schiel³

et al. 2015

Journal of Biotechnology

146

105

View full text Add to dashboard Cite

show abstract

“…Therefore, selection of targets with GC contents of about 50%-70%, and those with less or no base-pairing with the sgRNA sequence, is desirable. Use of target sequences with higher GC contents may potentially lead to a higher risk of off-targeting (Tsai et al, 2015), a critical issue in clinical research. However, for basic and applied research in plants, off-targeting may not be a critical problem, because the risk of off-targeting in plants by the CRISPR/Cas9 system may not be higher than that of the frequent somatic mutations that occur during the tissue culture-based transformation or other mutagenesis treatments.…”

Section: Discussionmentioning

confidence: 99%

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

et al. 2015

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CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

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“…Off-target analysis was performed based on the GUIDE-seq methodology (52). The workflow was described previously (40).…”

Section: Data Availabilitymentioning

confidence: 99%

Clinically Relevant Gene Editing in Hematopoietic Stem Cells for the Treatment of Pyruvate Kinase Deficiency Hemolytic Anemia

Baquero

Quintana-Bustamante

Dever

et al. 2021

Preprint

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Pyruvate Kinase Deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene, which constitutes the main cause of chronic non-spherocytic hemolytic anemia. PKD incidence is estimated in 1 in 20,000 people worldwide. The PKLR gene encodes for the erythroid pyruvate kinase protein (RPK) implicated in the last step of the anaerobic glycolysis in red blood cells. The defective enzyme fails to maintain normal erythrocyte ATP levels, producing severe hemolytic anemia, and can be fatal in severe patients. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cells (HSPC) transplantation, so far. However, HSPC transplant is associated with a significant morbidity and mortality, especially in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR/Cas9 system and rAAVs for donor matrix delivery to build an efficient and safe system to knock-in a therapeutic donor at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient recipient mice. Moreover, erythroid cells derived from edited PKD-HSPCs restored normal levels of ATP, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene editing strategy may represent a lifelong therapy to restore RPK functionality in RBCs of patients and correct PKD.Single Sentence SummaryClinically relevant gene editing in hematopoietic stem cells for the treatment of Pyruvate Kinase Deficiency.

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689. Defining Genome-Wide Off-Target Cleavage Profiles of CRISPR-Cas RNA-Guided Nucleases Using GUIDE-Seq

Cited by 3 publications

References 0 publications

Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity

Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants

Clinically Relevant Gene Editing in Hematopoietic Stem Cells for the Treatment of Pyruvate Kinase Deficiency Hemolytic Anemia

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