Current trends in gene recovery mediated by the CRISPR-Cas system

Abstract

The CRISPR-Cas system has undoubtedly revolutionized the genome editing field, enabling targeted gene disruption, regulation, and recovery in a guide RNA-specific manner. In this review, we focus on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders. Through the action of DNA repair mechanisms, CRISPR-mediated DNA cleavage at a genomic target can shift the reading frame to correct abnormal frameshifts, whereas DNA cleavage at two sites, which can induce large deletions or inversions, can correct structural abnormalities in DNA. Homology-mediated or homology-independent gene recovery strategies that require donor DNAs have been developed and widely applied to precisely correct mutated sequences in genes of interest. In contrast to the DNA cleavage-mediated gene correction methods listed above, base-editing tools enable base conversion in the absence of donor DNAs. In addition, CRISPR-associated transposases have been harnessed to generate a targeted knockin, and prime editors have been developed to edit tens of nucleotides in cells. Here, we introduce currently developed gene recovery strategies and discuss the pros and cons of each.

Fig. 1. Schematic of the cell’s own repair processes.

They include the non-homologous end joining (NHEJ) pathway, the microhomology-mediated end joining (MMEJ) pathway, and the homology-directed repair (HDR) pathway.

Fig. 2. Gene recovery strategies in the absence of donor DNA.

a Gene recovery methods with one guide RNA. b Gene recovery methods with dual guide RNAs. PTC premature termination codon.

Fig. 3. Gene recovery strategies involving donor DNAs.

a HDR-mediated gene correction rescue methods. b Homology-independent gene recovery methods. LHA left homology arm, RHA right homology arm, LTR long terminal repeat.

Fig. 4. Gene recovery strategies in the absence of DNA DSB generation.

a Schematic of a cytosine base editor (CBE), an adenine base editor (ABE). b Schematic of the insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) with a type I transposon-associated CRISPR-Cas system and of RNA-guided DNA insertion with a CRISPR-associated transposase (CAST). LE transposon left end, RE transposon right end. c Schematic of a prime editor (PE) and its working mechanism. Reverse transcriptases in PEs copy the information in the pegRNAs into DNA target sites.