CRISPR/Cas9 gene editing has emerged as a powerful tool for making genomic sequence changes. However, concerns surrounding off-target effects and undesired alterations in the genome have been raised. To address these challenges, a mutant form of Cas9 known as dCas9 (deficient Cas9 or Dead Cas9) has been developed. Unlike Cas9, dCas9 lacks the ability to cleave double-stranded DNA but can still be guided to specific genomic sites by specific RNA molecules called gRNA.
The fusion of dCas9 with various functional domains allows for precise epigenomic modifications at targeted loci, enabling the modulation of gene expression without altering the underlying DNA sequence. This CRISPR-based gene expression regulation offers several possibilities, including increasing gene expression through linked transcriptional activators (known as CRISPRa), repressing expression through linked methyltransferases for methylation (referred to as CRISPRi), introducing genomic base changes using linked APOBEC enzymes, visualizing specific genomic loci in real-time through fluorescence markers attached to tagged dCas9, and carrying out specific enzymatic reactions at targeted loci using linked enzymes.
One such tool is the “dCas9-Domain” fusion, which has proven to be invaluable for epigenomic analysis. Gentarget, a leading biotechnology company specialized lentivirus products, has developed premade lentivirus products to facilitate various applications in this field. These products have made tasks such as expression repression via KRAB, promoter methylation using different methyltransferases, genomic base changes via APOBEC, genomic base repair through reverse transcriptase, genomic frame shifting using DNA polymerase, expression activation via VP64-SunTag, p65, HSF1, GAL4, and other epigenomic modifications via different domains, easier than ever before.
By leveraging CRISPR-based epigenomic expression regulation, researchers can delve deeper into the complexities of gene regulation and gain a better understanding of how genes are controlled and influenced. We will explore the various applications and advancements in this exciting field, shedding light on the potential of CRISPR technology for epigenomic studies.