CRISPR/Cas9 Gene Editing

Nuclease-based genome editing tools can induce mutagenesis faster and more economically than traditional ES cell gene targeting techniques. Therefore, nuclease-based tools are more accessible to the scientific community.

However, despite the impressive results obtained so far, these tools are still limited by significant shortcomings such as low reliability and low flexibility in model design, transgene size and off-target effects. Such deficiencies mean that the use of robust ES cell gene targeting methods is still preferred for sophisticated and complex model creation.

genOway's strategy is to use the most appropriate technique (nuclease-based genome editing or ES cell-based gene targeting) in terms of time and risk for each project.

CRISPR/Cas9 Structure and Mechanisms of Action

A CRISPR/Cas9 nuclease system requires two components: a Cas enzyme for cutting the target sequence and a single guide RNA (sgRNA), which binds to the target sequence of 20-base pair (bp). The target sequence (complementary to the sgRNA sequence) is followed by two cytosine nucleotides because the sgRNA binds best when the opposite DNA strand is comprised of any nucleotide followed by two guanines (-NGG). This sequence is called a Protospacer Adjacent Motif (PAM) sequence. The PAM varies depending on the origin of Cas9.

CRISPR/Cas9 creates specific double-strand breaks at the target locus that trigger DNA repair mechanisms. These corrections result in two types of genome modifications: constitutive Knockouts (KO) through non-homologous end joining and Knockins (KI) through homologous recombination.

• A CRISPR/Cas9 system is a simple single-base pairing recognition system that requires a short PAM sequence downstream from the target sequence.
• Customized CRIPSR/Cas9 nuclease design is the most simple of the different nucleases (e.g. TALEN, ZNF).

Published data and our internal data (see paragraphs 3 and 4) clearly demonstrate that sequence composition is a key factor in determining the efficacy and specificity of in vivo CRISPR/Cas9 action.

CRISPR/Cas9 Efficacy

Design simplicity does not guarantee high efficacy. The literature shows that in eukaryotic cells and rodents, efficacy is highly variable from one target sequence to another.

Since 2013, genOway has been heavily investing in CRISPR/Cas9 nuclease design and protocol optimization in order to obtain high efficacy. The following parameters were critical:

• Genomic region structure
• Low predicted off-target effects
• Injection protocols

• Some CRISPR/Cas9 nucleases have very low and sometimes barely detectable activity. The technology’s quality and performance may be high, but it should not be oversold or overestimated. The technology is not yet fully reliable.
• genOway has optimized sequence design and improved injection protocols to achieve high CRISPR/Cas9 efficacy.

CRISPR/Cas9 Off-Target Activity

One major drawback of this nuclease technology is the non-specific (off-target) cutting of genomic sequences. Such "additional" mutations in the genome may strongly affect the phenotype of the generated model.

As for transgenic animals (random insertion), it is highly recommended to analyze several independent lines of mutant animals to statistically demonstrate the relationship between the genetic modification and the observed phenotype.

CRISPR/Cas9 off-target events can be explained by two different molecular mechanisms:

• Cas9 nuclease targets one sequence, but slightly different sequences (degenerated sequences) may also be recognized and cut. Such off-target mechanisms can be investigated by sequencing the degenerated target sequences.
• Cas9 nuclease could randomly cut within the genome: A molecular mechanism has been suggested. In this mechanism, when the nuclease complex scans DNA for specific sequences, random cutting can occur. This off-target activity can only be measured through whole genome sequencing.

High off-target activity has been demonstrated for ZFNs and TALENs. CRISPR/Cas9 nuclease systems also produce off-target effects, but fewer than with ZFNs and TALENs. However, it is still too early to determine if this difference is statistically significant.

All models developed by genOway are systematically validated for minimal off-target activity. Our CRISPR/Cas9 nuclease design is rigorous to minimize off-target effects (see 'genOway's ongoing R&D programs', below).

Genetic Background Used with CRISPR/Cas9 Nucleases

Most laboratories use outbred or hybrid mouse lines (e.g., FVB, B6D2) for their experiments since they provide robust experimental conditions (e.g., more embryos, high post injection survival, more pups obtained per embryo injection).

Nevertheless, a genetically modified model is usually more scientifically valuable in an inbred background. genOway has developed its CRISPR/Cas9 platform using C57BL6 genetic backgrounds.

genOway has extensive experience applying CRISPR/Cas9 nuclease technology to C57BL6 genetic backgrounds using protocols and procedures adapted to work with this fragile genetic background.

For rat models, genOway has built a standard offer using Sprague-Dawley (SD). Other genetic backgrounds are under development.

Constitutive Knockout Models: Mutation Types Created by CRISPR/Cas9 Nucleases

Using nucleases for KO model development is based on the NHEJ (non-homologous end joining) mechanism that creates a mutation in the target sequence (see Nuclease Mechanisms of Action).

CRISPR/Cas9 nucleases create small mutations within the target sequence. These small mutations provide more reliable constitutive KO model development (design and creation) than with TALENs or ZFNs.

Knockin Models: Mutation Types Created by CRISPR/Cas9 Nucleases

Using nucleases to create Knockin (KI) models is a major objective. The scientific community faces two special challenges:

• KI model design requires precise insertion positioning. Consequently, the design flexibility of the target sequence is limited.
• Nuclease-mediated homologous recombination events occur much less frequently than nuclease-mediated mutations. Therefore, nuclease efficacy must be high.

Nevertheless, very promising results have already been obtained using CRISPR/Cas9 nucleases to create KIs using small DNA fragments or transgenes (Ref. 25; genOway results Figure 7a, b and 8a, b).

Figure 7a. CRISPR/Cas9 Knockin strategy using small DNA fragments harboring loxP site (background: C57BL6) A) Description of the endogenous loci with LoxP site and recombined loci with the digestion site. B) Homologous Recombination (HR) event detection by PCR followed by digestion. C) PCR products were sequenced to confirm Knockin by HR. D) Experimental data.	Figure 7b. CRISPR/Cas9 Knockin strategy using ss oligonucleotide harboring a point mutation (background: C57BL6) A) Description of the endogenous and recombined loci. B) PCR products were sequenced to identify KI event. C) Experimental data.
Figure 8a, b. Transgene insertions into two cytokine genes using CRISPR/Cas9 (background: C57BL6)

genOway's Ongoing R&D Programs

a) Multi-Targeting Models Using CRISPR/Cas9

Multi-targeting (mutating several targets in the same embryo or cell) is feasible with CRISPR/Cas9 nuclease technology.
Publications describe simultaneous targeting of two to five genes in mice (Ref. 7, 14, 25), in rats (Ref. 20, 22) and in cells (Ref. 4).
The success rate varies and depends on the efficacy of each CRISPR/Cas9.

Figure 9.
genOway has tested protocols to efficiently target multiple sites in the murine genome. From our experience, the key parameter is the relative efficacy of the different CRISPR/Cas9 used.

b) Large Deletions Using CRISPR/Cas9

c) CRISPR/Cas9 Off-Target Effects

We have several ongoing programs that focus on off-target effects:

• Determine procedures and protocols to efficiently detect off-target effects on sequences similar to the targeted sequence (degenerate sequences) as well as on non-related sequences (random cutting).
• Reduce off-target activity by 1) applying more stringent rules for CRISPR/Cas9 nuclease design and 2) optimizing injection protocols for reduced nuclease activity (quality and duration).