Editing the Genome Without Double-Stranded DNA Breaks

Abstract

Genome editing methods have commonly relied on the initial introduction of double-stranded DNA breaks (DSBs), resulting in stochastic insertions, deletions, and translocations at the target genomic locus. To achieve gene correction, these methods typically require the introduction of exogenous DNA repair templates and low-efficiency homologous recombination processes. In this perspective we describe alternative, mechanistically motivated strategies to perform chemistry on the genome of unmodified cells without introducing DSBs. One such strategy, base editing, uses chemical and biological insights to directly and permanently convert one target base pair to another. Despite its recent introduction, base editing has already enabled a number of new capabilities and applications in the genome editing community. We summarize these advances here and discuss the new possibilities that this method has unveiled, concluding with a brief analysis of future prospects for genome and transcriptome editing without double-stranded DNA cleavage.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

“…With relatively good agreement, they suggest that correcting CFTR in 5-15% of cells should restore Cl − secretion to near wild-type levels, editing activity within the range achieved in this study. Additional challenges remain to attain a therapeutic end, including the methods for codelivery of homologous recombination templates, the identification of alternate CRISPR nucleases or base editors 64,65 , and evaluation in larger animal models 38 .…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…The recent observation that Cas9 is capable of infrequently inducing larger genomic rearrangements stimulated significant apprehension . Such events may be cell type‐specific and could be preventable by using genome‐editing approaches that do not induce double‐strand breaks, such as nickases or base editors …”

“…108 Such events may be cell type-specific and could be preventable by using genomeediting approaches that do not induce double-strand breaks, such as nickases 109,110 or base editors. 111 To minimize the incidence of OTEs and immune responses, it is critical to minimize the dose and lifetime of CRISPR editing reagents. Lentiviral administration involves gene integration, which promotes persisting expression of the Cas9 enzyme, although this can be minimized using integration-deficient lentivirus.…”

CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors

“…However, those approaches act transiently and have nonspecific effects for the drugs. Alternatively, CRISPRmediated homology-directed repair (HDR) can be used for gene correction, but is limited by low correction efficiency, especially in differentiated non-replicating cells from higher eukaryotes including humans 8,9,10 .…”