Base editing and prime editing in laboratory animals

Abstract

Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple ‘search and replace’ editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.

Application of CRISPR/Cas9 System in Establishing Large Animal Models

“…The basic principle is to use nCas9 to nick the non-target strand at the target location, and then use reverse transcriptase to generate the required sequence using the template RNA. Then the FEN1 endonuclease is able to excise the sequence called flap during this process and contributes to the genome repairing ( Anzalone et al, 2019 ; Caso and Davies, 2022 ). Although this technique has only been reported in the establishment of small animal models ( Liu et al, 2020 ), it is of great significance for the study of diseases caused by various base mutations in large animal models because it can mediate almost all types of base conversions.…”

Prime Editing: An All-Rounder for Genome Editing

“…In this case, the 3′ flap can be retained and the other can be discarded. Later, the overhang containing the edit integrated into the desired site through a cellular DNA repair system [ 9 , 18 ].…”

“…Researchers are currently most concerned about the editing efficiency of PE, which is criticized for its low efficiency at specific loci or certain types of editing compared with BE [ 18 , 26 ]. According to a seminal article reporting PE, scientists have been trying to improve its efficiency.…”

Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases

“…Base editing is being used to study and treat hereditary diseases in a variety of cell types ( Zeng et al, 2020 ; Tekel et al, 2021 ) and organisms ( Kim et al, 2021c ), including animal models of human hereditary diseases ( Liu et al, 2018 ; Caso and Davies, 2022 ). Alternatively, some preclinical experiments demonstrated the prospects of using the base editing technologies to treat diseases.…”