Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs

An, Liyou; Hu, Yeshu; Chang, Shiwei; Ling, Pingping; Zhang, Fenli; Liu, Jiao; Liu, Yanhong; Chen, Yexiang; Yang, Lan; Presicce, Giorgio Antonio; Du, Fuliang

doi:10.1038/srep25199

Cited by 10 publications

(8 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 18 previously published studies studies that have targeted individual loci in the mouse genome with single sgRNAs have not specifically addressed the symmetry of deletions 10 11 17 18 31 32 33 34 35 36 37 38 39 40 41 42 43 44 . Deleted sequences were available from seven studies 11 18 31 34 39 43 44 ( Supplementary Data 1 ), but only one 18 showed large enough data sets to permit a direct comparison with ours. Kim and colleagues 18 targeted two genomic sites based on 84 founders and with a cutoff of two-fold, 82% of the deletions were asymmetric compared to 73% in our study with 139 mice representing nine genomic sites ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

et al. 2017

View full text Add to dashboard Cite

Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

show abstract

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The picture emerging from studies in animal systems is similar to that in plants: most on‐target and off‐target mutations reported in animals are short deletions of up to 40 bp (An et al ., ; Friedland et al ., ; Kim et al ., ). Although less common, deletions as large as 250 bp have been reported in mice (Heckl et al ., ) and human cells (Liang et al ., ), but insertions are usually shorter, typically 1–15 bp (Cheng et al ., ; Friedland et al ., ; Kim et al ., ; Liang et al ., ).…”

Section: Mutation Signaturesmentioning

confidence: 99%

Patterns of CRISPR/Cas9 activity in plants, animals and microbes

Bortesi

Zhu

Zischewski

et al. 2016

Plant Biotechnology Journal

131

View full text Add to dashboard Cite

SummaryThe CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.

show abstract

“…However, many factors are associated with sgRNA activity and specificity. Various experimental methods can be used to detect sgRNA activity and specificity, and one is to truncate the length of CRISPR sgRNA [30][31][32].…”

Section: Discussionmentioning

confidence: 99%

“…A previous study showed that tru-gRNAs with 17-19 nts spacer are more sensitive to mismatches than those with length of 20 nts, which can effectively reduce off-target mutations [30]. Meanwhile, tru-gRNAs with 18 complementary nts and Cas9 nucleases can effectively generate gene knockout mice with a significantly high efficiency in a site-dependent manner [32]. However, the use of tru-gRNAs can reduce CRISPR/Cas9-mediated genome editing activity in a cell type-dependent manner [33,34].…”

Section: Ivyspringmentioning

confidence: 99%

Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity

et al. 2019

View full text Add to dashboard Cite

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is effective for genome editing and now widely used in life science research. However, the key factors determining its editing efficiency and off-target cleavage activity for single-guide RNA (sgRNA) are poorly documented. Here, we systematically evaluated the effects of sgRNA length on genome editing efficiency and specificity. Results showed that sgRNA 5′-end lengths can alter genome editing activity. Although the number of predicted off-target sites significantly increased after sgRNA length truncation, sgRNAs with different lengths were highly specific. Because only a few predicted off-targets had detectable cleavage activity as determined by Target capture sequencing (TargetSeq). Interestingly, > 20% of the predicted off-targets contained microsatellites for selected sgRNAs targeting the dystrophin gene, which can produce genomic instability and interfere with accurate assessment of off-target cleavage activity. We found that sgRNA activity and specificity can be sensitively detected by TargetSeq in combination with in silico prediction. Checking whether the onand off-targets contain microsatellites is necessary to improve the accuracy of analyzing the efficiency of genome editing. Our research provides new features and novel strategies for the accurate assessment of CRISPR sgRNA activity and specificity.

show abstract

Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs

Cited by 10 publications

References 35 publications

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

Patterns of CRISPR/Cas9 activity in plants, animals and microbes

Evaluation of the effects of sequence length and microsatellite instability on single-guide RNA activity and specificity

Contact Info

Product

Resources

About