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Review
. 2023 Apr 7;4(1):10.
doi: 10.1186/s43556-023-00115-5.

Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications

Lifang Zhou  1 , Shaohua Yao  2
Affiliations
Review

Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications

Lifang Zhou et al. Mol Biomed. .

Abstract

Recently, clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 derived editing tools had significantly improved our ability to make desired changes in the genome. Wild-type Cas9 protein recognizes the target genomic loci and induced local double strand breaks (DSBs) in the guidance of small RNA molecule. In mammalian cells, the DSBs are mainly repaired by endogenous non-homologous end joining (NHEJ) pathway, which is error prone and results in the formation of indels. The indels can be harnessed to interrupt gene coding sequences or regulation elements. The DSBs can also be fixed by homology directed repair (HDR) pathway to introduce desired changes, such as base substitution and fragment insertion, when proper donor templates are provided, albeit in a less efficient manner. Besides making DSBs, Cas9 protein can be mutated to serve as a DNA binding platform to recruit functional modulators to the target loci, performing local transcriptional regulation, epigenetic remolding, base editing or prime editing. These Cas9 derived editing tools, especially base editors and prime editors, can introduce precise changes into the target loci at a single-base resolution and in an efficient and irreversible manner. Such features make these editing tools very promising for therapeutic applications. This review focuses on the evolution and mechanisms of CRISPR-Cas9 derived editing tools and their applications in the field of gene therapy.

Keywords: Base editing; CRISPR/Cas9; Gene Delivery; Gene therapy; Prime editing.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Structure of spCas9/sgRNA/DNA complex. a Overall structure of spCas9/sgRNA/DNA complex, with PI, HNH and RuvC domains shown in yellow, green and blue respectively. b and c Detailed structure showing the interaction between PI domain and PAM (b), with key amino acids highlighted in pink (c). d Detailed structure showing the HNH domain, RuvC domain and target DNA, with key amino acids (D10 and H840) shown in sphere. Mutation of Asparticacid 10 (D10) or Histidine 10 (H840) to Alanine (A) silences the RuvC or the HNH domain respectively, resulting in TS nickase or NTS nickase respectively. Mutation of both amino acids silences both domains, resulting in catalytic dead Cas9. e Detailed structure showing the key amino acids interaction with target strand in HNH domain. f Detailed structure showing the key amino acids interaction with target strand in RuvC domain
Fig. 2
Fig. 2
Editing processes outcomes of Cas9 nuclease, base editor and prime editor
Fig. 3
Fig. 3
CRISPR-associated transposon and recombination systems. a Cas transposases include both Cas proteins and transposase-associated components. Cargo DNA is identified by its left end (LE) and right end (RE) sequences and bound by transposase proteins (Tns). Guide RNA binding to Cas nuclease brings transposase to the specific site, and transposase integrates the DNA cargo into the target site. The target site is duplicated and flanks the integrated LE–cargo–RE sequence. Each Cas-transposase complex has a specific requirement for the spacer length of guide RNA and a unique position preference of integration site. b reassembly of guide RNA-programmed recCas9 at the target sites
Fig. 4
Fig. 4
Therapeutic Strategies of Cas9 nuclease, base editor and prime editor. Different Cas9-related editing tools (above) can produce different types of edits to the genome (middle) and thus can cause different changes to the corresponding genes (below)
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