Precision Medicine, CRISPR, and Genome Engineering Moving from Association to Biology and Therapeutics

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4.4 A New Era of Animal Model Production

by the CRISPR/Cas9 Technology

Soon after the discovery of the profound genome-editing potential of the CRISPR/

Cas9 system in human cell line studies [ 6 – 8 ], Rudolf Jaenisch’s group tested the

system in mouse zygotes and was able to show that targeted mutant mice can be

generated with a previously unattainable speed and efficiency [ 9 , 10 ]. One microin-

jection procedure delivering CRISPR/Cas9 into mouse zygotes can directly edit the

mouse genome. The injected zygotes are subsequently transferred to recipients and

give rise to mice with intended mutations at a high frequency [ 5 ]. The types of

genome modification mediated by CRISPR/Cas9 include, but are not limited to,

genetic alterations in single or multiple loci, large DNA inversions and deletions,

point mutations, targeted knock-ins, reporters, and conditional alleles. The CRISPR/

Cas9 method omits the use of embryonic stem cells and dramatically reduces the

time needed for generation of a new mouse model. The simplicity and efficiency of

the CRISPR system has triggered a revolution in the way the research animals are

made.

Our facility, the Transgenic Animal and Genome Editing Core in Cincinnati

Children’s Hospital Medical Center, was established in 1994 and since that time has

provided all of the classical transgenic services, such as pronuclear injection, blas-

tocyst injection, and sperm and embryo cryopreservation and recovery. In early

2014, we incorporated the CRISPR/Cas9 genome engineering capacity into the

facility, as inspired by the work of the Jaenisch group [ 9 , 10 ]. Since then, we have

provided our customers with a comprehensive genome-editing service, beginning

with a design to achieve their desired mutation, construction of a CRISPR/Cas9

editing system, and, ultimately, production of genotype-confirmed founder animals.

In 3 years, we, as a mid-sized transgenic facility, have generated 164 rodent models

by CRISPR, including 63 knockouts/large deletions/large inversions, 72 small

knock-ins using donor oligos, 12 large knock-ins using donor plasmids, and 17

conditional alleles, averaging one project per week. Compared to the conventional

embryonic stem cell targeting approach, the remarkable effectiveness of the

CRISPR/Cas9 technology on mutant rodent production is clearly evident. In addi-

tion to these rodent models, we have completed a dozen cell editing projects in

human Induced Pluripotent Stem (iPS) cells, mouse ES cells, and cancer cell lines.

The success of the CRISPR/Cas9 genome-editing experiments depends on the

choice of guide RNA. Guide RNA can be either a two-RNA (crRNA and tracrRNA)

composition or single-guide RNA (sgRNA; a chimeric RNA that combines both

crRNA and tracrRNA). Similar to other programmable nucleases (e.g. ZFNs and

TALENs), the performance of the CRISPR/Cas9 system is affected by two major

parameters: on-target activity and specificity. Although all the initial publications

demonstrated the high efficiency of the CRISPR/Cas9-mediated genome editing in

rodent zygotes [ 9 – 14 ], we quickly realized that not all sgRNAs work. About 10% of

sgRNAs (4 in the first 40 sgRNAs we injected) failed to produce any editing at the

target loci. Given that any unsuccessful targeting is costly, we established a reliable

C.L. Yuan and Y.-C. Hu