Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease

Qin, Wenning; Dion, Stephanie; Kutny, Peter M.; Zhang, Yingfan; Cheng, Albert W.; Jillette, Nathaniel; Malhotra, Ankit; Geurts, Aron M.; Chen, Yi-Guang; Wang, Haoyi

doi:10.1534/genetics.115.176594

Cited by 240 publications

(193 citation statements)

References 19 publications

Supporting

Mentioning

189

Contrasting

Unclassified

Order By: Relevance

“…Some progress has been made in this area, including the use of a smaller Cas9 ortholog [159], which is more amenable to packaging into viral vectors. Other approaches are also being pursued, including non-viral methods for delivery of DNA or mRNA by nanoparticles [160] and electroporation [161], or direct delivery of Cas9 protein [162]. Another issue is the long-term effects of Cas9 expression in heterologous eukaryotic cells, which remain unexplored.…”

Section: Genome Editing Applicationsmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

545

460

View full text Add to dashboard Cite

Background: Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defence systems include receptor masking, restriction-modification (R-M systems), DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL) and abortive infection, all of which act in an innate, non-specific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems, i.e. clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR (pre-cr) RNAs and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference). Advances:A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed significantly to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPRCas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes.The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.Outlook: Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be done. About the evolution of CRISPR-Cas systems, the continuing discovery of novel CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario. The recent discovery and characterization of new CRISPR-Cas types with many unique features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related variants. Hence, newly discovered CRISPR-Cas systems need to be thoroughly dissected with the aforementioned multi-disciplinary approaches to gain insight in their biological role, to unravel their molecular mechanism, and to harness their potential for biotechnology. As to the biology, key outstanding questions include the ecological roles of microbial ...

show abstract

Section: Genome Editing Applicationsmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

545

460

View full text Add to dashboard Cite

show abstract

“…In the pioneering stage of applications of the CRISPR-Cas9 system, various physical and non-viral delivery approaches (Figure 5), such as electroporation 34 , nanoparticles 28 and hydrodynamic injection 35 have been used to deliver CRISPR-Cas9 to target cells. Although viral vectors are more efficiently to deliver nucleic acids, such as the plasmid-based CRISPR/Cas9, safety is the primary advantage of non-viral vectors.…”

Section: Physical and Non-viral Delivery Of Crispr-cas9mentioning

confidence: 99%

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

Liu

zhang

Líu

et al. 2017

Journal of Controlled Release

408

399

View full text Add to dashboard Cite

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes.

show abstract

“…A second round of confirmatory experiments, performed on a gene-by-gene approach, could confirm the gene identification process. The rate-limiting step for such a CRISPR-based genetic screening in mice is oocyte microinjection, which could be replaced by oocyte electroporation (Hashimoto & Takemoto 2015, Qin et al 2015, Takahashi et al 2015.…”

Section: Crispr/cas9 and The Future Of Endocrinologymentioning

confidence: 99%

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

Markossian¹,

Flamant²

2016

Journal of Molecular Endocrinology

View full text Add to dashboard Cite

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.

show abstract

Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease

Cited by 240 publications

References 19 publications

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

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

Contact Info

Product

Resources

About