It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Researchers review how two such experiments—the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology—have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. They highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.
Despite the obvious advances that have been made as a result of iPSC and editing technologies, several challenges remain. A key limitation remains that human cells prefer to choose the imprecise NHEJ pathway to repair a DSB rather than use the more precise homologous DNA repair pathway using an exogenous repair template. Due to this pathway choice, editing events often result in NHEJ-mediated insertions and deletions at the DSB rather than the intended homology-mediated modification. NHEJ-mediated gene disruption can be useful when the researcher or clinician intends to generate a loss-of-function event. However, in most clinical treatment settings the generation of a defined allele with high frequency will be essential to devise treatment options that require editing to result in gain of function at endogenous genes. Approaches to shift the balance away from NHEJ and toward homology-mediated repair included inhibiting NHEJ with small molecules or controlling the timing of CRISPR/Cas9 delivery with respect to the cell-cycle stage. These approaches are promising, yet we are currently far away from testing the efficacy of treatment strategies that rely on gene repair or gain-of-function approaches using high-frequency HR repair events of endogenous genes.
Facing this challenge, recent studies used creative ways to take advantage of NHEJ-meditated genome editing and the fact that the simultaneous expression of two nucleases can meditate the excision or inversion of the sequence internal to the two SSNs. In the specific case of Duchenne muscular dystrophy, Cas9 was employed to excise 725 kb of genomic sequences, which removed a premature STOP codon in the disease-causing DMD gene and thereby restored the reading frame and partial protein function.
Similarly, Cas9-mediated genome editing in patient-specific iPSCs was used to genetically correct the disease-causing chromosomal inversions found in patients with Hemophilia A, demonstrating that NHEJ-based approaches can be used to model and correct large-scale genomic alterations underlying human disease.
Elegant work that also takes advantage of the fact that genomic sequences between two SSN cuts can reinsert back into the locus in an inverted manner recently demonstrated that CTCF sites interact with each other in an orientation-dependent manner. Using this approach Guo et al. elucidate the impact of the directionality of CTCF sites in the mediation of large-scale genome interactions and transcriptional regulation.
Another challenge of genome editing in human cells is that human cells have relatively short conversion tracts. This means that even when a DSB is repaired by homology-directed repair (HDR) and not the NHEJ machinery, modifications can only be made with reasonable frequency very close to one side of the DSB. This presents a major obstacle toward the introduction of complex genetic changes in hPSCs. The use of Cpf1, a class 2 CRISPR effector that uses the same basic principles as Cas9, but cleaves DNA further away from the PAM sequence and generates a single-stranded overhang, may help increase the rate of HDR over NHEJ events (Zetsche et al., 2015). Overcoming this challenge will significantly facilitate the engineering of human stem cells, as it will allow us to refine the human genome more efficiently. Eventually this could result in similar resources that have been used in yeast and mESCs, such as a comprehensive collection of conditional human knockout iPSC libraries, with a homozygous iPSC line for each human gene carrying an exon flanked by LoxP sites.