No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Iyer, Vivek; Boroviak, Katharina; Thomas, Mark; Doe, Brendan; Riva, Laura; Ryder, Edward; Adams, David J.

doi:10.1371/journal.pgen.1007503

Cited by 114 publications

(64 citation statements)

References 16 publications

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“…Our results are consistent with previous results in zebrafish demonstrating limited off-target activity at select predicted regions (Hruscha et al, 2013; Varshney et al, 2015) and with recent work in mice that found limited support for off-target effects genome-wide (Iyer et al, 2018). However, we are limited to detecting potential off-target variation within the exon-capture space of the genome.…”

Section: Discussionsupporting

confidence: 93%

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Mooney

Davis

Katsanis

2019

Preprint

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1Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 2 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-3 target events. While recent work in rodents outlines favorable conditions for specific editing and 4 uses a trio design to control for the contribution of natural genome variation, the potential for 5 CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we 6 performed whole exome sequencing (WES) on two generations of offspring derived from the 7 same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first 8 generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation 9 (F1). We did not observe an increase in the number of transmissible variants in edited 10 individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not 11 precipitate an inflation of deleterious point mutations. 12 Results 34 Generating and sequencing CRISPR-Cas9-edited F0 and F1 individuals 35We focused on three different genes (anln, kmt2d, and smchd1) for which (a) we have 36 substantial experience in this model organism and (b) which give reproducible, quantitative 37 defects in kidney morphogenesis (Hall et al., 2018), mandibular and neuronal development 38 (Tsai et al., 2018), and craniofacial morphogenesis (Shaw et al., 2017). For each locus, we used 39 sgRNAs that had the following three characteristics. First, for each of the three genes, we 40 selected an sgRNA with demonstrated high efficiency (100%) and an sgRNA with low efficiency 41 (~30%), as determined by heteroduplex analysis and Sanger sequencing of cloned PCR 42 products (Hall et al., 2018; Shaw et al., 2017; Tsai et al., 2018) (Suppl. Figure 1). Second, we 43 mandated that all sgRNAs have a high specificity score (MIT specificity score 79-99 for each 44 sgRNA; Suppl. Table S1). Finally, we required that each sgRNA was predicted to generate off-45 target effects with low cutting frequency determination (CFD) scores (mean = 0.17, range = 0-46 0.73; Suppl. Figure 2). 47 289

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Section: Discussionsupporting

confidence: 93%

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Mooney

Davis

Katsanis

2019

Preprint

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“…However, the extensive characterisation of the target loci that ONT sequencing supports, in both founder and subsequent generations, does not suffice to validate newly mutated lines. Indeed, checks of off-target events (Anderson et al, 2018;Iyer et al, 2018), in particular, those physically linked to the locus of interest) and copy counting of the donor sequence by ddPCR to eliminate additional integrations remain essential and complementary steps to fully validate G1 animals (Supplemental Figures S3 and S4 and Codner et al, 2018).…”

Section: A Simple Processmentioning

confidence: 99%

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Codner

Allan

et al. 2019

Preprint

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Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

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“…Along those lines, the original study on WGS of CRISPR/Cas9 edited mice suggested the presence of extensive off-target mutations (14), which was, however, likely the result of an imperfect experimental design as pointed out in editorials (15)(16)(17)(18)(19). Further WGS investigations by Iyer and colleagues as well as our group (20,21) using trio studies demonstrated that CRISPR/Cas9 does not introduce an excess of off-target mutations. Therefore, it is prudent to examine critical issues of base editing, such as the extent of off-target mutations with a larger number of mice and under several conditions.…”

Section: Introductionmentioning

confidence: 95%

Cytosine but not adenine base editor generates mutations in mice

Lee

Smith

Liu

et al. 2019

Preprint

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Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls.

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No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

Cited by 114 publications

References 16 publications

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Cytosine but not adenine base editor generates mutations in mice

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