Simple and rapid design of genetically engineered rodent models.

Customized mouse and rat model generation via the nuclease-based system CRISPR/Cas9 outcompetes traditional methods in terms of time. Additionally, CRISPRs can be designed to target virtually all genes in a eukaryotic genome, just by synthesis of short sequence specific RNAs.

Like TALENs, the CRISPR/Cas9 system (for review see Doudna and Charpentier, 2014 and Hsu et al., 2014 ) is used to introduce double strand breaks into a genome in a sequence specific manner. Whereas TALENs use a combination of hard-to-assemble protein domains recognizing the specific DNA sequence, the CRISPR/Cas9 system makes use of easily synthesized small RNA molecules that are applicable to a wider veriety of target sequences. These small RNAs, originally derived from Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in bacteria, target the nuclease Cas9 to the specific genomic region and introduce double strand breaks (Jinek et al., 2012 ).

The introduction of double strand breaks leads to an error prone repair mechanism (non-homologous end joining, NHEJ) and to insertions or deletions (INDELs) at its position which can result in functional knockout of a gene (left). As the efficiency of the CRISPR/Cas9 system is much better than for TALENs, it is also possible to introduce small knock-ins by adding templates for homologous recombination, although the mechanism of homologous recombination has a much a lower efficiency than NHEJ (right).

As compared to TALENs and other nuclease based techniques, CRISPR/Cas9 is the perfect system in terms of cutting efficiency. However, as the higher efficiency may lead to a higher number of off-target cutting for CRISPR/Cas9, a thorough analysis of off-targets is highly recommended.

CRISPR/Cas9 rodent model generation possibilities at PolyGene:

CRISPR Knockout mice and rats
Microinjections will start within a month after inception of the project, cutting the total animal model generation time to 4-5 month (half of the time as compared to standard knockout generation via ES cell targeting). The resulting genetically modified animals are screened via sequencing for changes in the gene of interest. Optionally, PolyGene will carry out a thorough sequence analysis of potential off-target effects in the genome.
At PolyGene, CRISPR-based knockouts have all but replaced the traditional way of generating constitutive knockout mouse models (as well as knockout rat models).
For CRISPR/Cas9 microinjection into the oocyte, cf. Wang et al., 2013, Parikh et al., 2015, Paquet et al., 2016 for examples in mouse.

CRISPR Knock-in mice and rats
PolyGene offers the introduction of point mutations and smaller insertions (e.g. eGFP tags) at competitive pricing. The precedure is similar to the generation of CRISPR knockout rodent models, but includes the coinjection of specially purified oligos or small donor sequences, which carry the point mutation(s) of interest.
Larger insertions are more challenging and require complex strategies, e.g. when requiring coinjection of upstream and downstream guide RNAs, or targeting less permissive loci. In this instances, we will elaborate a careful alternative locus targeting strategy with the costumer.
For larger insertions, some groups have reported good efficiency for some “easy to targeted” loci like Rosa26 (Chu et al., 2016 ), but other genes have proven to be more difficult to target with homologous recombination in oocytes.
CRISPR Gene Targeting in ES cells
CRISPR targeting via homologous recombination in ES cells (Kraft et al., 2015) can indeed be a good alternative for genes where direct injection into oocytes was not successful, the desired changes are too complex or assay in ES cells are desired. In these instances CRISPR-based targeting can be superior if a selection of ES cells, as necessary in traditional approaches, has to be circumvented.
Doudna JA, Charpentier E.
The new frontier of genome engineering with CRISPR-Cas9.
Science. 2014;346(6213):1258096

Hsu PD, Lander ES, Zhang F.
Development and applications of CRISPR-Cas9 for genome engineering.
Cell. 2014;157(6):1262-78.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E.
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science. 2012;337(6096):816-21.

Wang H, Yang H, Shivalila C, Dawlaty M, Cheng AW, Zhang F, Jaenisch R.
One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering.
Cell. 2013; 153(4): 910–918.

Parikh BA, Beckman DL, Patel SJ, White JM, Yokoyama WM.
Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.
PLoS One. 2015;10(1):e0116484.

Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, Olsen KM, Gregg A, Noggle S, Tessier-Lavigne M.
Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.
Nature. 2016;533(7601):125-9.

Chu VT, Weber T, Graf R, Sommermann T, Petsch K, Sack U, Volchkov P, Rajewsky K, Kühn R.
Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.
BMC Biotechnol. 2016;16:4.

Kraft K, Geuer S, Will AJ, Chan WL, Paliou C, Borschiwer M, Harabula I, Wittler L, Franke M, Ibrahim DM, Kragesteen BK, Spielmann M, Mundlos S, Lupiáñez DG, Andrey G.
Deletions, Inversions, Duplications: Engineering of Structural Variants using CRISPR/Cas in Mice.
Cell Rep. 2015 pii: S2211-1247(15)00029-7.

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