Questioning unexpected CRISPR off-target mutations in vivo

S, Kim; J, Park; D, Kim; K, Kim; Bae, Sejong; Schlesner, Matthias; J, Kim

doi:10.1101/157925

Cited by 10 publications

(9 citation statements)

References 13 publications

(10 reference statements)

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“…Lastly, we identified a heritable T to A substitution which led to a single nucleotide variation (SNV) and here results in a premature stop codon ( Figure 3B and 3C ). This occurs when a single bp deletion is followed by a single bp insertion, an event very rarely observed for CRISPR/Cas9 ( Kim et al 2017 ). In conclusion, the pDE-Cas9 vectors allow for efficient and inheritable genome editing in Arabidopsis with the possibility of producing transgene free homo-allelic mutants in the T2 generation.…”

Section: Resultsmentioning

confidence: 99%

A Dual sgRNA Approach for Functional Genomics inArabidopsis thaliana

Pauwels

Clercq

Goossens

et al. 2018

G3 Genes|Genomes|Genetics

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Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.

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Section: Resultsmentioning

confidence: 99%

A Dual sgRNA Approach for Functional Genomics inArabidopsis thaliana

Pauwels

Clercq

Goossens

et al. 2018

G3 Genes|Genomes|Genetics

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“…Our statistical test, however, indicates a significant difference between the heterozygosity of them (p-value=0; Supplementary Notes), proving that the overall derived mutation pattern in Study_1 is not germline-like. Note that Lareau et al and Kim et al 4,6 recently demonstrated that the two CRISPR-Cas9 treated mice (F03, F05) in Study_1 are actually more closely related to each other genetically than to the control mouse, proving that these mutations are most likely preexisting variants. Our analysis result further presented that there are unusual SNVs arose in Study_1 combined with pre-existing variants, and the overall mutation pattern in vivo are not germline-like.…”

Section: Snvsmentioning

confidence: 93%

Unexpected CRISPR off-target mutation patternin vivoare not typically germline-like

Wei

Chuai

et al. 2017

Preprint

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To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

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“…79 In addition to the viewpoint from Lareau et al, there are some other researchers questioning Schaefer et al's point of view. Kim et al 80 and Wilson et al, 81 for instance, both think that the observed variants in Schaefer et al's experiments are much more proper to be explained by the differences in the genetic background of the mice rather than mutations caused by Cas9. Thus, this issue is currently under drastic controversy and more efforts are needed to clarify it.…”

Section: Off-target Effect and Solutionsmentioning

confidence: 97%

CRISPR/Cas9-mediated noncoding RNA editing in human cancers

et al. 2017

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Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

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Questioning unexpected CRISPR off-target mutations in vivo

Cited by 10 publications

References 13 publications

A Dual sgRNA Approach for Functional Genomics inArabidopsis thaliana

A Dual sgRNA Approach for Functional Genomics inArabidopsis thaliana

Unexpected CRISPR off-target mutation patternin vivoare not typically germline-like

CRISPR/Cas9-mediated noncoding RNA editing in human cancers

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