The CRISPR/Cas9 Nuclease System for KO & KI Model Creation

1. CRISPR/Cas9 Nuclease System Structure

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 structure

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.


2. CRISPR/Cas9 Nuclease System Design

Designing a CRISPR/Cas9 nuclease system is less complicated than for Transcription Activator-Like Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs). A CRISPR/Cas9 system is based on simple, single bp recognition whereas TALENs and ZFNs are multimeric proteins formed by 17 or 8 protein domains (TALEs or ZFs). Each TALE or ZF recognizes one or three bps respectively (Figures 2 and 3). Consequently, designing an efficient and reliable TALEN or ZFN is difficult and labor-intensive. Even experienced laboratories must create and test several TALENs or ZFNs before validating one.

TALE Nuclease structureZinc-Fnger-Nuclease structure

  • 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.


3. CRISPR/Cas9 Nuclease 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

CRISPR-Cas9 efficacy table

Figure 4. 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describing the efficacy of 97 different CRISPR/Cas9 designs in rodent or cellular systems and compared them with the unpublished data we obtained from 88 different CRISPR/Cas9 designs.


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.


4. CRISPR/Cas9 Nuclease 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.

CRISPR-Cas9 off-target quantification

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


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 8. genOway's ongoing R&D programs, below).


5. 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.

CRISPR-Cas9 on C57BL6 background

Figure 6.
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.

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 Spraque-Dawley (SD). Other genetic backgrounds are under development.


6. 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).

Size of CRISPR-Cas9-induced mutations

Figure 7.
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.


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.


7. 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 8a, b and 9a, b).

Oligonucleotide Knockin by CRISPR-Cas9 harboring LoxP site Oligonucleotide Knockin by CRISPR-Cas9 harboring point mutation
Figure 8a.
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 8b.
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 9a, b.
Transgene insertions into two cytokine genes using CRISPR/Cas9 (background: C57BL6)

Transgene Knockin by CRISPR-Cas9 nucleaseTransgene Knockin by CISPR-Cas9 Ires LacZ


8. 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.

CRISPR-Cas9 multi-targeting

Figure 10.
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

large deletion by CRISPR-Cas9

Figure 11.
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).



1) Mali P, Esvelt KM, Church GM
Cas9 as a versatile tool for engineering biology.
Nat Methods. 2013

2) Wei C, Liu J, Yu Z, Zhang B, Gao G, Jiao R
TALEN or Cas9 - rapid, efficient and specific choices for genome modifications.
J Genet Genomics. 2013

3) Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD
Genome editing with engineered zinc finger nucleases.
Nat Rev Genet. 2010

4) Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM
RNA-guided human genome engineering via Cas9.
Science. 2013

5) Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F
Multiplex genome engineering using CRISPR/Cas systems.
Science. 2013

6) Cho SW1, Kim S, Kim JM, Kim JS
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.
Nat Biotechnol. 2013

7) Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
Cell. 2013

8) Cradick TJ, Fine EJ, Antico CJ, Bao G
CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.
Nucleic Acids Res. 2013

9) Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F
DNA targeting specificity of RNA-guided Cas9 nucleases.
Nat Biotechnol. 2013

10) Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
Cell. 2013

11) Yang L, Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang PY, Daley G, Church G
Optimization of scarless human stem cell genome editing.
Nucleic Acids Res. 2013

12) Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J
Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.
Cell. 2014

13) Zhou J, Wang J, Shen B, Chen L, Su Y, Yang J, Zhang W, Tian X, Huang X
Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting.
FEBS J. 2014

14) Fujii W1, Kawasaki K, Sugiura K, Naito K
Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.
Nucleic Acids Res. 2013

15) Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M
Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.
Nat Biotechnol. 2013

16) Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, Choi JH, Ban YH, Ha SJ, Kim CH, Lee HW, Kim JS
Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases.
Genome Res. 2014

17) Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M
Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.
Sci Rep. 2013

18) Zhou J, Shen B, Zhang W, Wang J, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng Q, Huang X
One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering.
Int J Biochem Cell Biol. 2014

19) Ma Y, Zhang X, Shen B, Lu Y, Chen W, Ma J, Bai L, Huang X, Zhang L
Generating rats with conditional alleles using CRISPR/Cas9.
Cell Res. 2014

20) Li W, Teng F, Li T, Zhou Q
Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems.
Nat Biotechnol. 2013

21) Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I
Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.
Sci Rep. 2014

22) Ma Y, Shen B, Zhang X, Lu Y, Chen W, Ma J, Huang X, Zhang L
Heritable multiplex genetic engineering in rats using CRISPR/Cas9.
PLoS One. 2014

23) Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X
Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.
Cell Res. 2013

24) Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA
Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.
Proc Natl Acad Sci U S A. 2013

25) Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.
Cell. 2013

26) Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.
Nat Biotechnol. 2013

27) Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Nat Biotechnol. 2013

28) Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN
Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.
Science. 2014

29) Fujii W, Onuma A, Sugiura K, Naito K
One-step generation of phenotype-expressing triple-knockout mice with heritable mutated alleles by the CRISPR/Cas9 system.
J Reprod Dev. 2014

30) Zhong H, Chen Y, Li Y, Chen R, Mardon G
CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes.
Sci Rep. 2015

31) 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