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Generation of Knockout, Knock-in, and Humanized Mouse Models Using the CRISPR/Cas9 Technology: Lessons Learned and Open QuestionsSteffen Güttler1, Alexander Klimke1, Petric Kuballa1, Gesa Glöckel1, Jeanette Keßler1, Heidrun Kern1, Kenneth Albrecht2, Adriano Flora1, Eleanor Kolossovski2, Jochen Welcker1

1Taconic Biosciences GmbH, Cologne, Germany; 2Taconic Biosciences Inc., Rensselaer, NY

The CRISPR/Cas9 genome editing system has established itself as a versatile technology for inducing precise genetic alterations in a number of

different species, and has proved to have the potential to increase the efficiency and speed of producing genetically engineered models of human

disease. At Taconic Biosciences, we use both an in vivo strategy utilizing one-cell embryos and a complementary in vitro strategy utilizing embryonic

stem (ES) cells to generate both mouse and rat models with CRISPR/Cas9. We provide a broad data-set to illustrate our experiences using these two

strategies within our production pipeline.

The major advantages of in vivo gene editing using CRISPR/Cas-based methods are the significantly reduced time frame and effort involved in

establishing new mouse or rat models and the ability to utilize almost any available mouse and rat strain. The relative simplicity of this method compared

to ES cell-mediated approaches facilitates the generation of founder animals with reduced costs and effort, providing an attractive alternative to

homologous recombination-based approaches. We have implemented CRISPR/Cas9 technology in vitro for the accelerated generation of knockout

and simple knock-in (e.g. point mutations and small tags) mice and rats and successfully generated more than 200 models in the past three years.

However, the genetic modifications that can be introduced in the genome by the in vivo approach are currently limited to relatively simple allelic

configuration, such as single base substitutions, gene deletion, and insertion of short sequences. To overcome this limitation, we have combined the

use of CRISPR/Cas9 technology with the advantages of utilizing large targeting constructs in ES cells. The combination of the two technologies allows

for the generation of large and complex alleles with the precision and efficiency provided by the CRISPR/Cas system and, importantly, the humanization

of specific loci in the mouse genome by gene replacement to create relevant model for preclinical drug testing.

Advantages of Humanization by Genomic Replacement

• Successful means that the humanized allele has an expression of asimilar level (60% or more) of the endogenous mouse gene and thesame pattern of expression as measured by RT-qPCR.

• While Minigene Insertions and Open Reading Frame (ORF) Exchangesare easy to accomplish, the lack of regulatory elements within intronicregions often compromises the expression levels and patterns.

• We recommend genomic replacements as these seem to resembleexpression-levels and patterns most closely.

Protocol for CRISPR-mediated Gene Editing in ES Cells

Transfection of ES Cells to Introduce a Targeted Mutation via CRISPR/Cas9-mediated Gene Editing

ES cells (e.g. C57BL/6NTac or BALB/c) were grown on a mitotically inactivated feeder layer comprised of mouse embryonic fibroblasts in ES cell culture medium. Cas9, the specific gRNAs, and the targeting vector were co-transfected into cells along with a plasmid for the expression of the respective selection cassette. One day post transfection the antibiotic was transiently added to the medium to select for transfected cells. ES cell clones were isolated as soon they show a distinct morphology and were analyzed by PCR in a primary screen for recombination at the 5' and 3' side. Homologous recombinant ES cell clones were expanded and frozen in liquid nitrogen after extensive molecular validation by PCR and southern blot analysis.

Analysis of Off-target Effects• For all CRISPR embryo and CRISPR in ES cell projects, off-target

predictions were performed according to the number and positionof mis-matches within the gRNA sequence.

• For 12 CRISPR in ES cell projects and 43 CRISPR in embryo projects,potential loci were analyzed by PCR and sequencing.

• Within 505 loci of 31 ES cell clones and 310 loci of 555 G1-mice, onlythree off-target effects were detected:

— The first off-target in embryo with two deviations in the seed andone deviation in the non-seed region.

— The second off-target in ES cells with two deviations in the seed and one deviation in the non-seed region.

— The third off-target in embryo with no deviation in the seed and two deviations in the non-seed region (only present in one out of three validated clones).

• The off-target frequency with our setup is smaller than 0.4% andlower than the spontaneous mutation rate detected per generationor passage.

Protocol for CRISPR-mediated Gene Editing in Embryo

Pronuclear Injection

After administration of hormones, superovulated C57BL/6NJ females were mated with C5BL/6NJ males. One-cell stage fertilized embryos were isolated from the oviducts at dpc 0.5. For microinjection, the one-cell stage embryos were placed in a drop of M2 medium under mineral oil. A microinjection pipette with an approximate internal diameter of 0.5 micrometer (at tip) was used to inject the mixed nucleotide preparation into the pronucleus of each embryo. For knockout projects, the mix contained two specific gRNA and the Cas9-protein. For knock-in projects, an oligonucleotide was injected together with the appropriate gRNA. After recovery, 25–35 injected one-cell stage embryos were transferred to one of the oviducts of 0.5 dpc, pseudo-pregnant NMRI females.

Founder Analysis

Founder animals were genotyped for presence of the deletion or the inserted point mutation (and inserted restriction site). PCR samples per founder were subcloned and up to four clones were analyzed by DNA-sequencing.

Gene Knockout (KO) in Embryo Knock-in (KI) of a Point Mutation

in Embryo

Humanizations and Knock-in in ES cells

Life Born Rates of 43 Knock-out Projects

60

50

40

30

20

10

0

103 Injections (43 Projects)

Pu

p N

um

ber

Life Born Rates of 39 Knock-in Projects

98 Injections (39 Projects)

40

30

20

10

0

Pu

p N

um

ber

wt %indel %del%

CRISPR-mediated KO Approach: Percentage of Modifications in Embryo

103 Injections (43 Projects)

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

Perc

en

tag

e o

f T

ota

lG

en

oty

ped

An

imals

CRISPR-mediated KI Approach: Percentage of Modifications in Embryo

Perc

en

tag

e o

f T

ota

lG

en

oty

ped

An

imals

98 Injections (39 Projects)

wt %indel %del%

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

HeterozygousTargeted Clones Homozygous

17.8 kb (wt)

3.0 kb (hum)

wt 1 2 3 4 5

12.2 kb (hum)

5.9 kb (control)

PCR Pre-screen PCR Pre-screen

CRISPR-mediated KO Approach: Modifications in Embryo in Relation to Deletion Size

Size of Deletion [kb]

Perc

en

tag

e o

f T

ota

lG

en

oty

ped

An

imals

wt %indel %del%

00

01

01

01

01

01

01

01

02

02

02

02

03

03

03

03

03

03

04

04

04

05

05

06

06

06

07

07

08

09

10

10

12

18

19

24

59

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

90 105 120 150 180

Percentage of Knock-out Projects Completed within a Given Timeframe

Time for Completion [Days]

120%

100%

80%

60%

40%

20%

0%

Perc

en

tag

e o

f P

roje

cts

Co

mp

lete

d

KO 2014/2015KO 2016

90 105 120 150 180

Percentage of Knock-in Projects Completed within a Given Timeframe

Time for Completion [Days]

120%

100%

80%

60%

40%

20%

0%

Perc

en

tag

e o

f P

roje

cts

Co

mp

lete

d

KI 2014/2015KI 2016

CRISPR-mediated Tag-KI Approach: Percentage of Modifications in Embryo

Size of Insertion [nt]

100%

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

Perc

en

tag

e o

f T

ota

lG

en

oty

ped

An

imals

42 42 42 51 51 51 67 67 67

wt %indel %HDR %

CRISPR-mediated KI Approach: Percentage of Modifications in ES cells

Nu

mb

er

of

Valid

ate

d C

lon

es

Size of Insertion/Humanization [kb]

10

9

8

7

6

5

4

3

2

1

0

validatedheterozygous clone

validatedhomozygousclone

0.4 0.9 1 2.2 3.4 3.4 3.4 3.4 4.5 5 5.2 5.6 6.5 8.4 19

0 2 4 6 8 10 12 14 16 18 20

Homologous Recombination Rate of 15 Humanization and Knock-in Projects

Size of Insertion/Humanization [kb]

Perc

en

tag

e o

f H

om

olo

go

us

Reco

mb

inati

on

1.0%

0.8%

0.6%

0.4%

0.2%

0.0%

Nu

mb

er

of

pro

jects

!

"

#!

#"

$!

$"

%!

%"

&!

&"

Genomic Replacement

Minigene Insertion

ORF Exchange

Point Mutations

Exon Swapping

'())*++,(-

./+())*++,(-

Genomic Replacement

Minigene Insertion

ORF Exchange

Point Mutations

Exon Swapping

45

40

35

30

25

20

15

10

5

0

Genomic Replacement

Minigene Insertion

ORF Exchange

Humanizing Point Mutations

Exon Swapping

• CRISPR/Cas9 is a powerful tool that can speed up model generationfrom simple point mutations to large knock-ins and humanizations.

• While we can predictably control the design of each model, theefficiency of CRISPR/Cas9 strongly depends on the selected gRNA.

• Despite various prediction tool, the relation between gRNA-sequenceand cleavage-efficiency remains largely unknown.

• Off target effects are a controllable risk in our setup.

CONCLUSION

Improvements of procedures have led to higher reproducibility and overall efficiency. This resulted in a vast majority of projects completed in or ahead of the anticipated timeline.

Deletion efficiency is independent of the size of the deletion Tag sequences of up to 67 nucleotides were inserted using long oligonucleotides

Deletion efficiency is highly variable and dependent on gRNA sequence Homologous recombination efficiency is highly variable and dependent on the gRNA sequence

• Homologous recombination efficiency is highly variable, depending on thegRNA sequence

• We successfully inserted up to 45 kb of human sequence

CRISPR-mediated homologous recombination in ES cells occurs on both alleles with a high frequency

Mouse Genomic Locus

Mouse Genomic Locus

Targeted Allele (after CRISPR/Cas9-medicated Gene Editing)

Mouse Genomic Locus

Constitutive KI-PM Allele (after CRISPR/Cas9-medicated Gene Editing)

Constitutive Humanized Allele (after CRISPR/Cas9-medicated Gene Editing)

Homologous Recombination Rate of 15 Humanization and Knock-in Projects

Success of Humanization Projects Completed at Taconic Biosciences

CRISPR-mediated KI Approach: Percentage of Modifications in ES cells

PCR Pre-screen Southern Blot Analysis

Nu

mb

er

of

pro

jects

!

"

#!

#"

$!

$"

%!

%"

&!

&"

Genomic Replacement

MinigeneInsertion

ORF Exchange

Point Mutations

Exon Swapping

'())*++,(-

./+())*++,(-

Genomic Replacement

MinigeneInsertion

ORF Exchange

Point Mutations

Exon Swapping

45

40

35

30

25

20

15

10

5

0

Genomic Replacement

Minigene Insertion

ORF Exchange

Humanizing Point Mutations

Exon Swapping

Life Born Rates of 43 Knockout Projects Life Born Rates of 39 Knock-in Projects

CRISPR-mediated KO Approach: Percentage of Modifications in Embryo CRISPR-mediated KI Approach: Percentage of Modifications in Embryo

CRISPR-mediated KO Approach: Modifications in Embryo in Relation to Deletion Size

CRISPR-mediated Tag-KI Approach: Percentage of Modifications in Embryo

Percentage of Knockout Projects Completed within a Given Timeframe Percentage of Knock-in Projects Completed within a Given Timeframe

wt %

indel%

del%

wt %

indel%

del%

validated heterozygous clone

validated homozygous clone

wt %

indel%

HDR%

wt %

indel%

del %

KO 2014/2015

KO 2016

KI 2014/2015

KI 2016

successful

unsuccessful