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The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.

Since the initial reports describing CRISPR-Cas9, labs across the globe have leveraged this valuable gene editing tool to alter the genomes of living cells. With the goal of generating more precise and efficient genome changes, scientists and engineers have mutated, evolved, and covalently altered Cas9 in order to predictably edit the genetic code. Here, we highlight recent advancements and contributions to the growing field of Cas9 engineering. We present key aspects of Cas9 engineering efforts focused on sgRNA manipulation, PAM-recognition, specificity, deaminase fusions, reverse-transcriptase fusions, and structural rearrangements of this important gene-modifying tool.