19Cas9 methods were used to remove the premature stop codon in the DMD gene
leading to Duchenne Muscular Dystrophy and resulted in partial restoration of pro-
tein function [ 180 ]. Additionally, patient-specific iPSCs generated from Hemophilia
A patients were used in conjunction with Cas9-mediated editing to remedy the
large-scale chromosomal inversions that underlie the disease process [ 181 ].
As for the future of iPSC and CRISPR therapy to treat human disease, manychallenges remain. In the clinical setting, treatments generally rely on producing a
defined gain of function at desired genes with high frequency; with this method,
however, human cells prefer the imprecise pathway of non-homology end joining
(NHEJ) repair of the double strand breaks in DNA as opposed to the homology-
mediated editing [ 182 ]. Therefore, many approaches have been taken to shift the
DSB repair pathway from the generation of NHEJ-mediated insertions and dele-
tions to homology-mediated repair; these include cell-cycle dependent control of
CRISPR/Cas9 delivery via small molecular NHEJ-inhibitors [ 183 – 186 ].
Additionally, the goal of generating complex hiPSCs with a wide variety of genetic
alterations is hindered by the short conversion tracts of human cells and resulting
limitation of either NHEJ or HDR mechanism to one side of the DSB [ 187 ]. This
poses the biggest challenge of broadly applying iPSCs and CRISPR/Cas9 to edit-
ing the human genome as well as reveals the unrealized potential of the technology
to produce tremendously helpful resources, such as condition human knockout
iPSC libraries.
Fig. 1.3 Schematic of isolation of somatic cells from patients, generation of induced pluripotent
stem cells, correction of disease causing traits, differentiation into specific cell type and transplan-
tation back into the host
1 Viral Vectors, Engineered Cells and the CRISPR Revolution