Advancement in research using CRISPR/Cas9 technology has revealed the potential of CRISPR to traverse beyond simple applications of gene editing. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) are two such applications that reveal the gene-regulatory power of the CRISPR technology.
Conventionally, CRISPR/Cas9 system utilises Cas9 endonuclease to create DSB in the target DNA with the help of sgRNA. However, CRISPRi and CRISPRa utilise dCas9 (also known as endonuclease deficient) which is incapable of generating DSB. Rather dCas9 introduces RNA-directed transcriptional control.
In CRISPRi, dCas9 with/without transcriptional repressor domain (e.g., Krüppel-associated box (KRAB)), complexes with the sgRNA and is recruited to the transcription start site (TSS), to repress transcription. The dCas9-KRAB complex directs heterochromatin formation at the promoter site (usually immediately adjacent to the target gene) to reduce the target gene transcription.
On the other hand, in CRISPRa, dCas9 fuses with transcriptional activator and along with sgRNA are recruited to TSSs to initiate their overexpression. Recruiting multiple transcriptional activators to the TSS can increase the overexpression with a single sgRNA molecule.
Both CRISPRa and CRISPRi are capable of modulating gene expression transiently and operate at the transcription level. These methods enable scientists to understand the sensitivity of biological systems with respect to the gene expressions being modulated. Although CRISPRi is complementary to RNA interference (RNAi) method, it has lower sequence-specific off-target effects and is capable of modulating both coding and non-coding genes.
Thus, CRISPRi and CRISPRa add to the versatility of the CRISPR technology. In addition to manipulating gene, CRISPRi and CRISPRa can serve as genetic screens to study the role of specific genes in various diseases such as leukaemia and melanoma. Apart from their function as genetic screens, CRISPRa can drive endogenous gene expression withing living organisms and CRISPRi can be used to probe different cell lines. With further improvement in the design of sgRNA and dCas9 constructs, CRISPRi and CRISPRa technologies would pave the way for the development of precision medicine.