Limitations of CRISPR/Cas9Developed from a bacterial adaptive immune defense system, CRISPR/Cas9 allows the generation of null, conditional, point mutation, reporter, or tagged alleles at a much faster pace and lower cost compared to conventional methods that rely on homologous recombination in embryonic stem cells.
Our experience has shown that CRISPR/Cas9 is an efficient tool for the generation of simple alleles, such as constitutive knockout and knock-in of point mutations. However, this is not the technology of choice for the introduction of more complex modifications1,2,3,4,5, such as targeted insertion of transgenes, recombinases or reporters, all of which still rely on homologous recombination in embryonic stems cells.
Targeted editing of DNA using CRISPR/Cas9 requires the injection of three components into the (mouse) zygote: the Cas9 mRNA or protein, single-guide RNAs to specific to the gene target, and an oligonucleotide containing the DNA to be inserted. This approach induces double-stranded breaks (DSB) at the Cas9 cleavage site, triggering endogenous homology-directed repair (HDR) pathways to mediate the insertion of the template sequence into the target site using the oligonucleotide as a donor.
CRISPR's main limitations are the high frequency of random integration of the template DNA and the activation of the non-homologous end joining (NHEJ) DNA repair pathway, which induces microdeletions at the DSB site. This off-target and unintended modifications require founder animals be extensively analyzed to exclude the presence of aberrant alleles at the target site and random insertion of the template DNA in other genomic locations.
The Easi-CRISPR TechnologyThese technical challenges significantly limited the utility of CRISPR/Cas9 genome editing technology until the arrival of Easi-CRISPR. Published in Genome Biology in 2017, Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) is a novel and robust approach for the efficient generation of knock-in alleles in mice. The method was tested on different loci, and six knock-in lines were successfully generated with FlpO recombinase, the reverse tetracycline transactivator (rtTA), and the reporters mCherry and mCitrine with efficiencies ranging from 25% to 67%6.
Unlike existing methods, which predominately use double-stranded DNA or short single-stranded DNA (ssDNA) donors, Easi-CRISPR uses long ssDNA donors ranging from 500 base pairs to 2 kilobases to deliver targeted insertion at high frequency.
Advantages of Easi-CRISPREasi-CRISPR has two significant advantages: ssDNA molecules do not randomly integrate into the genome, and they serve as a template for the HDR with much higher efficiency than double-stranded DNA.
These advantages are rapidly positioning Easi-CRISPR as the method of choice to generate models that possess general or tissue-specific recombinases (e.g., Cre or Flp) or reporter genes such as EGFP or mKate2, since a cDNA inserted downstream of a tissue-specific promoter will be expressed with the same pattern as the endogenous target locus.
Moreover, it is possible to use Easi-CRISPR to insert a human cDNA within the first coding exon of its mouse orthologue, therefore generating a genetically humanized mouse model expressing the human instead of the mouse protein.
These types of models could only be efficiently generated by random-insertion transgenesis in zygotes or homologous recombination using a targeting construct in embryonic stem cells.
Limitations of Easi-CRISPRThe primary factor limiting the use of Easi-CRISPR is not the technology itself but rather the need to ensure a high-fidelity ssDNA as the template. Most ssDNA synthesis technologies rely on a transcription step followed by reverse transcription of the RNA into ssDNA using viral RNA-dependent DNA polymerases (reverse transcriptases). Since both the RNA polymerases and the reverse transcriptases usually lacking the proof-reading capabilities characteristic of the higher fidelity DNA-dependent DNA polymerases, the resulting ssDNA molecules derived from this process may carry mutations. If such an ssDNA template is used in Easi-CRISPR, the resulting allele will incorporate the secondary mutation, potentially compromising the resulting model.
Conditional Knockouts with Easi-CRISPRConditional knockouts are models where a gene of interest can be deleted in a tissue-specific manner. Due to their flexibility, conditional knockouts are frequently used in both basic and translational research. Despite initial optimism1, the standard CRISPR approach has proven quite challenging in the generation of conditional knockout alleles. Continued refinement of the Easi-CRISPR technology may allow many of these challenges to be overcome.
Taconic Biosciences' experience in the generation of conditional lines using this new approach has so far yielded mixed results. In general, we find the efficiency of the Easi-CRISPR technology for the introduction of conditional alleles to still be low. Much of this is due to the fact that conditional alleles require insertion of flanking palindromic sequences into the targeted loci. This is relatively easy when using the conventional approach of a targeted construct as both modifications are integrated into the construct and therefore "move" as one in the process. With CRISPR, insertion of the two palindromic sequences are independent events, and success is achieved only when both are inserted into the same allele of the target gene (i.e. in cis). The challenge becomes even greater when the desired insertion sites are separated by thousands of base pairs. In our experience using Easi-CRISPR to generate more complex conditional alleles, the resulting alleles carry several unintended modifications that can be attributed to multiple recombination sites and sister chromatid exchanges.
To further compound the challenges, the mosaic founder animals can carry various allelic combinations, making it almost impossible to figure out whether a procedure has been successful or not until the targeted allele is transmitted to the next generation via breeding. The implication is that success or failure of the generation procedure of a conditional knockout allele can be assessed only after two mouse generations, corresponding to roughly six months. Since the risk of failure is still quite high (over 50%), Taconic continues to recommend the use of standard targeting in ES cells to generate this kind of alleles (see table below) as the most time- and cost-efficient solution.
Despite the challenges when using Easi-CRISPR to generate conditional models, this new technology brings tremendous ease and speed of CRISPR to the generation of targeted "gain of function" type models such as targeted transgenics, tissue-specific recombinases and reporter models. We are confident that continued refinement CRISPR/Cas methodologies will eventually overcome some of the challenges regarding conditional models outlined above.
|Point Mutation Knock-in|
|Short Tag Knock-in|
Easi-CRISPR: What's Next?Compared to standard CRISPR, Easi-CRISPR allows the introduction of sophisticated alleles in the mouse and rat genome in a cost- and time-effective way. The development of new technologies allowing the generation of long ssDNA fragments with a low error rate will undoubtedly increase the number of alleles that can be generated using this approach.
At this point, Taconic has had positive experiences with the synthesis of molecules up to 3 kilobases, but it is conceivable that longer ssDNA templates will favor the generation of even more complex allelic configurations (e.g., humanizations by gene replacement rather than by insertion of a cDNA).
Taconic's scientists are also actively working on developing methodologies to allow for the reliable generation of conditional knockouts by inhibiting inter-chromosomal recombination events and improving the screening procedures to identify correctly targeted founder animals.