Fig. 1

The evolution of CRISPR tools that have been harnessed in the investigation of cancer biology. Since the inception of CRISPR-associated 9 (Cas9) gene editing in mammalian cells, there has been a rapid expansion in the field of CRISPR technology. This expansion has led to the development of various specialized CRISPR variants designed to tackle specific challenges. Scientists have created these variants through deliberate design and evolutionary processes, resulting in improved flexibility in recognizing protospacer adjacent motifs (PAMs) and increased precision in target selection. Additionally, they've harnessed naturally occurring variants from different bacterial species, like Cas12a (Cpf1) and Cas13, for effective combinatorial knockout (KO) and precise RNA targeting, respectively. To broaden the range of CRISPR applications, researchers have combined transcriptional effectors with catalytically inactive Cas9 (dCas9), allowing precise targeting of the transcriptome and epigenome. Furthermore, CRISPR base editing has enabled the introduction of specific transition mutations using a Cas9 nickase (Cas9n) fused with adenine or cytosine deaminase. In the case of cytosine base editing enzymes (BEs), they use a uracil glycosylase inhibitor (UGI) to prevent base excision repair and promote C > T transition mutations. A significant advancement known as prime editing has emerged, which involves fusing a dCas9 with a reverse transcriptase, enabling the engineering of various mutation types, such as missense mutations, insertions, and deletions. This is guided by a sequence template and an extended prime editing guide RNA (pegRNA). Additionally, to facilitate unbiased proteome mapping, researchers have employed engineered ascorbate peroxidase (APEX2) tethered to dCas9, enabling targeted biotinylation at specific genomic locations. Reprinted from [11] with permission from Springer Nature