Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

Abstract CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.

Download Full-text

Generation of genetically modified mice using SpCas9-NG engineered nuclease

Scientific Reports ◽

10.1038/s41598-019-49394-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Wataru Fujii ◽

Haruka Ito ◽

Takuya Kanke ◽

Arisa Ikeda ◽

Koji Sugiura ◽

...

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Knockout Mice ◽

High Efficiency ◽

Genetically Modified ◽

Protospacer Adjacent Motif ◽

Mouse Zygotes ◽

Genetically Modified Mice

Abstract Although genetically modified mice can be generated with high efficiency by using CRISPR/Cas9-mediated genome editing in mouse zygotes, only the loci with a protospacer-adjacent motif (PAM) sequence are targetable. The present study investigated the usability of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes. In addition to the 5′-NGG sequence, SpCas9-NG recognized the 5′-NGA, 5′-NGC and 5′-NGT sequences in mouse zygotes as PAMs that were appropriate for the generation of knockout mice. Moreover, SpCas9-NG-mediated genome editing enabled the generation of knock-in mice untargetable by the conventional SpCas9 in mouse zygotes. These results suggest that SpCas9-NG-mediated genome editing in zygotes is available for the generation of knockout and knock-in mice at the locus corresponding to NGN-PAM.

Download Full-text

An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Download Full-text

Genetically modified mouse models in studies of luteinising hormone action

Molecular and Cellular Endocrinology ◽

10.1016/j.mce.2006.03.026 ◽

2006 ◽

Vol 252 (1-2) ◽

pp. 126-135 ◽

Cited By ~ 27

Author(s):

Ilpo Huhtaniemi ◽

Petteri Ahtiainen ◽

Tomi Pakarainen ◽

Susana B. Rulli ◽

Fu-Ping Zhang ◽

...

Keyword(s):

Mouse Models ◽

Luteinising Hormone ◽

Genetically Modified ◽

Hormone Action

Download Full-text

Genetically modified mouse models to investigate thyroid development, function and growth

Best Practice & Research Clinical Endocrinology & Metabolism ◽

10.1016/j.beem.2018.03.007 ◽

2018 ◽

Vol 32 (3) ◽

pp. 241-256 ◽

Cited By ~ 2

Author(s):

C. Löf ◽

K. Patyra ◽

A. Kero ◽

J. Kero

Keyword(s):

Mouse Models ◽

Genetically Modified ◽

Thyroid Development

Download Full-text

The role of genetically modified mouse models in predictive toxicology

Predictive Toxicology in Drug Safety ◽

10.1017/cbo9780511779053.016 ◽

2011 ◽

pp. 269-283

Author(s):

Glenn H. Cantor

Keyword(s):

Mouse Models ◽

Genetically Modified ◽

Predictive Toxicology

Download Full-text

Enhanced Photocatalytic Reduction of Cr(VI) by Combined Magnetic TiO2-Based NFs and Ammonium Oxalate Hole Scavengers

Catalysts ◽

10.3390/catal9010072 ◽

2019 ◽

Vol 9 (1) ◽

pp. 72 ◽

Cited By ~ 4

Author(s):

Yin-Hsuan Chang ◽

Ming-Chung Wu

Keyword(s):

Organic Contaminants ◽

High Efficiency ◽

Ammonium Oxalate ◽

Reaction Rate Constant ◽

Electron Hole ◽

Tio2 Nanofibers ◽

Tio2 Photocatalysts ◽

Survival And Development ◽

Hole Scavenger ◽

Separation Behavior

Heavy metal pollution of wastewater with coexisting organic contaminants has become a serious threat to human survival and development. In particular, hexavalent chromium, which is released into industrial wastewater, is both toxic and carcinogenic. TiO2 photocatalysts have attracted much attention due to their potential photodegradation and photoreduction abilities. Though TiO2 demonstrates high photocatalytic performance, it is a difficult material to recycle after the photocatalytic reaction. Considering the secondary pollution caused by the photocatalysts, in this study we prepared Ag/Fe3O4/TiO2 nanofibers (NFs) that could be magnetically separated using hydrothermal synthesis, which was considered a benign and effective resolution. For the photocatalytic test, the removal of Cr(VI) was carried out by Ag/Fe3O4/TiO2 nanofibers combined with ammonium oxalate (AO). AO acted as a hole scavenger to enhance the electron-hole separation ability, thereby dramatically enhancing the photoreduction efficiency of Cr(VI). The reaction rate constant for Ag/Fe3O4/TiO2 NFs in the binary system reached 0.260 min−1, 6.95 times of that of Ag/Fe3O4/TiO2 NFs in a single system (0.038 min−1). The optimized Ag/Fe3O4/TiO2 NFs exhibited high efficiency and maintained their photoreduction efficiency at 90% with a recyclability of 87% after five cycles. Hence, taking into account the high magnetic separation behavior, Ag/Fe3O4/TiO2 NFs with a high recycling capability are a potential photocatalyst for wastewater treatment.

Download Full-text

Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽

10.3390/genes11060628 ◽

2020 ◽

Vol 11 (6) ◽

pp. 628

Author(s):

Elena McBeath ◽

Jan Parker-Thornburg ◽

Yuka Fujii ◽

Neeraj Aryal ◽

Chad Smith ◽

...

Keyword(s):

Genomic Dna ◽

Sanger Sequencing ◽

High Efficiency ◽

Genetically Engineered ◽

Rapid Evaluation ◽

Knock Out ◽

Guide Rna ◽

Guide Rnas ◽

Genetically Engineered Mice ◽

The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

Download Full-text