CRISPR/Cas9 gene editing

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 below) 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

Figure 1. CRISPR/Cas9 Cutting Efficacy
We compiled data from 20 peer-reviewed articles (Ref. 4-23) describing the efficacy of 97 different CRISPR/Cas9 designs in rodent or cellular systems and compared them with the unpublished data we obtained from 88 different CRISPR/Cas9 designs.

Results:

We achieved high cutting efficacy for 69% of our CRISPR/Cas9 designs.

• 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.

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Figure 2.
CRISPR/Cas9 off-target quantification is described in 18 recent publications. (Ref. 5, 7-10, 13-17, 19, 20, 22, 24-27)

Results:

Clear demonstration that off-target effects are present and can be very substantial.

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.

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Figure 3.
Eleven published CRISPR/Cas9 nuclease systems were tested in C57BL6 embryos (Ref. 14, 15, 21, 23, 28-31), while genOway has a cumulative experience of 97 designs that have been tested with success and validated in C57BL6 embryos.

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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.

Figure 4.
Most CRISPR/Cas9 nuclease-induced mutations are small mutations.

We have systematically analyzed the type of mutations generated by 10 different CRISPR/Cas9 designs in 121 mutants, produced by injection of Cas9 into C57BL6 zygotes.
The black vertical line represents the cutting site.
Grey dots represent deletions, green dots represent insertions and blue dots represent mismatches.

Results:

Seventy-six percent (76%) of all mutations are small mutations of fewer than 12 bp (68% small deletions and 8% small insertions).

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 5a, b and 8a, b).

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Figure 5a. 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.

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Figure 5b. 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.

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Figure 6a, 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.

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Figure 7.
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

Figure 8.
Genomic deletion using two CRISPR/Cas9 (background: C57BL6)

A) Description of endogenous and deleted loci.
B) Deletion was detected using primer spanning the sequence to be deleted (WT: 1261 bp; Deletion: 731 bp). PCR products were sequenced to confirm the deletion. Out of 9 samples, only samples 2 and 6 are showing the expected deletion.

Deletions vary from the expected size to several smaller ones.

We are currently developing protocols to efficiently and reliably delete gene sequences of up to 5 kb in length.

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).

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CRISPR-Cas9 patents: Don't get confused!

Why not?
Because there are only t​hree foundational patent portfolios!

To protect its clients from infringement risks, genOway invested substantially to hold licenses under all three portfolios.

Consequently:

• Our customers are authorized to use CRISPR-Cas9 models for R&D (internal or commercial) with no need to acquire any additional license from any third party
• You can acquire the authorization from genOway for the internal development or usage of rodent models from third-party providers

^{Each granted patent is represented by a colored dot.}

The three foundational patent portfolios in the eukaryotic field are from:

1. Sigma-Aldrich, aka Merck, which covers any CRISPR-Cas9 system for modifying a target DNA leading to a double-stranded break (this system may be associated with an exogenous sequence to perform Knockin)
2. Berkeley, which covers any CRISPR-Cas9 system for modifying a target sequence along with at least one guide RNA
3. Broad, which covers any CRISPR-Cas9 system for modifying a target sequence, where the CRISPR system is delivered to cells in the form of vectors