The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies

Verkuijl, Sebald A. N.; Rots, Marianne G.

doi:10.1016/j.copbio.2018.07.005

Cited by 111 publications

(89 citation statements)

References 34 publications

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“…A second possibility is that some key NHEJ repair enzymes required for CRISPR have higher expression in adults than in sporocysts or eggs; according to RNAseq metadata [25] this is the case for Smp_211060, previously identified [21] as a homolog of the Ku70/Ku80 genes that play a key role in NHEJ [47]. Finally, CRISPR might be more efficient in inducing mutations in SULT-OR in adults than sporocysts or eggs, because SULT-OR is expressed more in the former developmental stage, making its chromatin more open and therefore more accessible to the CRISPR machinery [19,[48][49][50].…”

Section: Discussionmentioning

confidence: 97%

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

Sankaranarayanan

Coghlan

Driguez

et al. 2020

Preprint

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At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a highquality annotated reference for Schistosoma mansoni. There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in S. mansoni, to produce somatic mutations in the omega-1 (ω1) gene. Here, we employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing SULT-OR. Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.

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Section: Discussionmentioning

confidence: 97%

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

Sankaranarayanan

Coghlan

Driguez

et al. 2020

Preprint

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“…These assays can detect potential off-targets with incredible sensitivity, but cannot provide information about how in vitro cleaved sites translate into bona-fide off-targets in cellular models. Chromatin state, modifications, other DNA-binding proteins, and collision with transcriptional complexes all influence Cas9 cutting efficiency and thus off-target mutagenesis in vivo ( 14 , 30 , 46 ). It is currently difficult to predict repair outcomes from in vitro data.…”

Section: Discussionmentioning

confidence: 99%

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Wienert

Wyman

Richardson

et al. 2018

Preprint

107

172

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21Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a 22 target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay 23 for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach 24 takes advantage of the recruitment of endogenous DNA repair factors for genome-wide 25 identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely 26 to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base 27 resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene 28 editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene 29 editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides 30 an unprecedented view of events that precede repair of the affected sites. 31 strengths, they also have certain weaknesses. Naïve prediction algorithms are for the most part 41 based on sequence similarity and currently have limited predictive power with very high false-42 positive rates (10). Assays that induce DSBs in vitro, such as Digenome-Seq (5), CIRCLE-Seq (6) 43 and SITE-Seq (7), have high sensitivity but dramatically under-or overestimate the number of 44 target sites that are actually modified in cellular models or in vivo (11). Nuclease concentration 45 within the cell (7), delivery method (ribonucleoprotein (RNP) vs. plasmid) (7, 12, 13) as well as 46 more complex cellular properties such as chromatin accessibility (14, 15) have been shown to 47

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“…Perhaps, the insertion of a long piece of DNA in a genomic region that has previously been edited (step 1) could be facilitated. It is known that chromatin state influences CRISPR-Cas9 editing efficiencies (Verkuijl and Rots 2018) and it is possible that the first cut makes the chromatin more accessible or sensitive for an subsequent cut. It any case, the cut required for the first step of Nested CRIPSR indicates that the chromatin in that region is accessible for Cas9 to perform the second and more limiting step.…”

Section: Universality Of Nested Crisprmentioning

confidence: 99%

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

Vicencio

Martínez-Fernández

Serrat

et al. 2018

Preprint

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CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient.The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors (≤200 bp) to insert 5' and 3' fragments in the place of interest. In the second step, we use these sequences as homology regions for Homology Directed Repair (HDR) with a dsDNA donor (PCR product, ≈700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.In the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Conveniently, we also found that both steps of Nested CRISPR could be performed in a single injection.Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.

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The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies

Cited by 111 publications

References 34 publications

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans

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