Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects

Shen, Bin; Zhang, Wensheng; Zhang, Jun; Zhou, Jianghua; Wang, Jian-Ying; Chen, Li; Wang, Lu; Hodgkins, Alex; Iyer, Vivek; Huang, Xingxu; Skarnes, William C.

doi:10.1038/nmeth.2857

Cited by 716 publications

(551 citation statements)

References 26 publications

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“…A way to drastically reduce the frequency of off-target effects is by using Cas9D10A, a Cas9 with an amino acid change that alters one of the two active sites of the nuclease. This turns Cas9D10A into a 'nickase', which only cuts one DNA strand (Mali et al 2013a, Ran et al 2013a, Shen et al 2014a). Cas9D10A nickase is not mutagenic, unless two sgRNAs are combined to target two opposite strands simultaneously, introducing two independent single-strand breaks at nearby sites (Shen et al 2014a).…”

Section: The Off-target Problemmentioning

confidence: 99%

“…This turns Cas9D10A into a 'nickase', which only cuts one DNA strand (Mali et al 2013a, Ran et al 2013a, Shen et al 2014a). Cas9D10A nickase is not mutagenic, unless two sgRNAs are combined to target two opposite strands simultaneously, introducing two independent single-strand breaks at nearby sites (Shen et al 2014a). In that case, the specificity of doublestranded DNA cleavage relies on the formation of two neighboring 17-20 nt RNA:DNA heteroduplexes instead of one.…”

Section: The Off-target Problemmentioning

confidence: 99%

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CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

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CRISPR/Cas9 is a recent development in genome editing which is becoming an indispensable element of the genetic toolbox in mice. It provides outstanding possibilities for targeted modification of the genome, and is often extremely efficient. There are currently two main limitations to in ovo genome editing in mice: the first is mosaicism, which is frequent in founder mice. The second is the difficulty to evaluate the advent of off-target mutations, which often imposes to wait for germline transmission to ensure genetic segregation between wanted and unwanted genetic mutations. However rapid progresses are made, suggesting that these difficulties can be overcome in the near future.

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Section: The Off-target Problemmentioning

confidence: 99%

Section: The Off-target Problemmentioning

confidence: 99%

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

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“…The introduction of TALEs and later CRISPRs as DNA recognition domains led to the decrease of the the sideeffects [32,33,45]. Engineering nicking enzymes in all kinds of the chimeric artificial nucleases also resulted in a substantial progress of this field [46][47][48][49][50][51][52]. Alternative nuclease domains have been applied in few cases in mainly monomeric chimera [53][54][55][56][57][58] and non-FokI-based zinc finger nucleases have also been developed by introducing mutations or small modifications into the zinc finger array, such as the exchange of the metal ion-binding cysteines to histidines [59][60][61].…”

Section: Current Artificial Nucleasesmentioning

confidence: 99%

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

Gyurcsik

2016

CPPS

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Artificial nucleases are designed for in vivo gene engineering, as the DNA cleavage performed at a specific target site enhances the effectiveness of the cell's DNA repair machinery. The therapeutic potential of the above phenomenon stems from the knowledge that (i) the shifted reading frame can be restored by non-homologous end-joining, or (ii) a DNA of erroneous sequence -causing a genetic disease -can be corrected by homologous recombination in the presence of a suitable DNA template. Besides the advantageous properties of the nowadays applied zinc finger nucleases, TALE nucleases and the CRISPR/Cas9 system, they possess a residual citotoxicity. This is related to offtarget cleavages, which could be prevented by the strict regulation of the enzymes. The studies on enzymes acting naturally in a controlled manner are beneficial to get better insight into their mechanism. Such enzymes or their appropriate domains may be the most promising alternatives to the presently applied ones. As an example, the DNA cleavage of the inactive HNH nuclease mutants is inducible in a multiple way. This property may be used for establishing a control mechanism and thus, in combination with specific DNA-binding domains they are good candidates for the catalytic site of artificial nucleases. Here we collect the results on the properties of the HNH nucleases that allow for their redesign into enzymes with possible therapeutic applications.

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“…Currently, there are a couple of techniques that scientists are using to improve target selectivity. First, Cas9 has two nuclease domains, namely, Cas9 HNH and Cas9 RuvC (Shen et al 2014). Researchers are currently looking into nullifying one of the nuclease sites followed by transformation of cells with two Cas9s; one with Cas9 HNH inactivated and the other with Cas9 RuvC.…”

Section: Future Directionsmentioning

confidence: 99%