Defense Arm of the Microbial Immune System
Being native to the bacterial immune system, no one imagined the immense potential of the CRISPR Cas system of the microbes. Limitations in the technology made it difficult for us to explore these proteins of tiny organisms. With the advent of sequencing technology in mid 2000 they discovered that bacteria and archaea has conserved repeat sequence called Clustered Regularly interspaced short palindromic repeat (CRISPR) and genes associated with CRISPR called Cas involved in the framework of bacterial acquired immune system protecting from phage by cleaving the phage DNA or RNA. The CRISPR-Cas system was classified into two major classes based on whether it involves single effector- Cas protein (Class 2) or multiple effector (Class 1).
A Leap from Defense System to Editing Technology
Endonuclease activity of CRISPR-Cas against foreign genetic material resembled RNAi. With the rise of the genome engineering era, where molecular scissors like Zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENS) were already becoming a breakthrough, spotlight was shed upon the CRISPR-Cas. Out of the two classes of CRISPR-Cas system, simplicity of Class II effector architecture made it attractive to be used in the battle of gene editing technology.
Variety Is The Spice Of Life
High Performance in the aspect of editing efficiency, High Precision, and ease of use of the technology made CRISPR-Cas popular. Cas variants belonging to different types of Class II like Cas 9 of type II, Cas 12 of type V and Cas 13 of type VI are being used now which expanded the application of technology. Besides, nucleases are now engineered to increase their performance in terms of specificity and the target scope. Sometimes their original role as endonuclease itself is modified like dCas9 (dead Cas 9) where the enzyme no longer has the nuclease activity but helps in targeting the editing machinery containing transcriptional activator or repressor to a precise region of the genome and alter the gene expression instead of gene modification, thus replacing the conventional siRNA technology which is used for knock in and knock out studies.
New Tools To The Toolbox
Though Cas and its variants improved the specificity and efficiency of gene editing, it suffered major setbacks in creating precise point mutations with low off target. Conventional CRISPR-Cas relied on the homology directed repair (HDR) by providing donor template, but it has extremely poor efficiency. To overcome this limitation, Liu and his group came up with a strategy where they are able to incorporate the precise mutation in donor independent fashion. They developed two enzymes which have the ability to convert one nucleotide to another nucleotide at targeted genomic coordinates. And it is called “Base editors” that includes adenine base editors (ABE) and cytidine base editors (CBE), which can mediate all the four transitions (C- >T, A- > G, T- > C, G- > A). Although 30% of pathogenic mutations comprises transition mutation, the majority are transversion mutations, but this cannot be performed by base editors. Very recently, a fusion protein of Cas nickase and reverse transcriptase domain called “Prime editors” was developed which can create all the 12 possible point mutations. Prime editors look like a very promising tool but still in the infant stage of development.
Gene editing technology is already making success in developing promising therapy, to be precise “permanent cure” for many diseases like hemophilia, sickle cell disease, retinitis pigmentosa and SCID. With the scientific advances in various fields like molecular biology, sequencing technology and bioinformatics, genome editing tools are also evolving, making us step into the new era of cure to the disease by rewriting the genetic code.
About The Author
Saranya Srinivasan is a first year Ph.D. student in the Molecular Immunology & Microbiology discipline of the Integrated Biomedical Sciences program (IBMS). She is working in Dr. Nu Zhang’s lab.
History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome. Editing Technology Yoshizumi Ishino, Mart Krupovic, Patrick Forterre Journal of Bacteriology Mar 2018, 200 (7) e00580-17; DOI: 10.1128/JB.00580-17
Anzalone, A.V., Koblan, L.W. & Liu, D.R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0561-9
“A Primetime For Gene Editing” https://firstname.lastname@example.org/a-primetime-for-gene-editing-6426a92f6003
TED “Can we cure genetic diseases by rewriting DNA? | David R. Liu”