CRISPR-Cas9 DNA Base-Editing and Prime-Editing

Abstract

Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.

Keywords: CRISPR/Cas9; adeno-associated vector; base-editing; gene therapy; genome engineering; prime-editing.

Figure 1

CRISPR DNA Base-Editing Tools. (A) DNA Base-editing. DNA base-editors encompass two key components: a Cas enzyme for programmable DNA binding and a single-stranded DNA modifying enzyme for targeted nucleotide alteration. Two classes of DNA base-editors have been described: cytosine base-editors and adenine base-editors. Cytosine deamination generates uracil, which base pairs as thymidine in DNA. Fusion of uracil DNA glycosylase inhibitor (UGI) inhibits the activity of uracil N-glycosylate (UNG), thus increasing the editing efficiency of cytosine base-editing in human cells. Adenosine deamination generates inosine, which has the same base pairing preferences as a guanosine in DNA. Collectively, cytosine and adenine base-editing can install all four transition mutations (C→T, T→C, A→G, and G→A). (B) Prime-editing. Prime-editors use an engineered reverse transcriptase fused to Cas9 nickase and a prime-editing guide RNA (pegRNA). The pegRNA contains the sequence complimentary to the target sites that directs nCas9 to its target sequence as well as an additional sequence spelling the desired sequence changes. Prime-editors expand the scope of DNA editing to not all transition and transversion mutations, as well as small insertion and deletion mutations.