CRISPR/CAS in the Forefront of Homology Directed Repair Mediated Genome Editing
Dr. Raviraj Banakar
University of Minnesota,
Deoxyribonucleic acid (DNA) carries genetic information essential for vital functioning of all living organisms, including some viruses. This information is faithfully inherited from one generation to another generation. Information in DNA composes of four letters, adenine (A), guanine (G), thymine (T) and cytosine (C), referred as nucleotides. For example, twenty-three pairs of human chromosomes contain 6.4 billion nucleotides, and twelve chromosomes of rice (Oryza sativa L) contain 4.3 billion nucleotides. Since the discovery of DNA double helix structure by James Watson and Francis Crick in 1953, several technologies were developed over the years which unravelled the role of DNA in maintaining the vital functions. Among these, technologies that enable opening up of specific segment of DNA in a living cell, cleave it at the site of interest so that the intended or desired modifications brought in are significant ones. This ability not only allows correcting nucleotides which were mutated/deleted over millions of years in an organism but also provides the unique opportunity to include new information at the site of interest. This is important because the difference between a cancerous and non-cancerous cell, and a herbicide susceptible Vs tolerant cultivars of a crop species can be traced back to variation in single or multiple nucleotides.
Hence, creation of targeted double strand breaks in DNA using sequence specific nucleases (SSN) is touted as the biggest leap in the history of molecular biology, as incorrect information that is inherited over millions of years can be corrected or new information can be added at specific sites. During the past several decades several SSN based technologies to create targeted double strand breaks (DSB) were developed which includes, meganucleases, zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALLEN), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) proteins. However, among these methods, CRISPR is easy to build, scale up, efficient and easy to adopt to different purposes.
Therefore, CRISPR is the method of choice for genome editing in many organisms, including humans and plants.
Once the DSB are created in cells by CRISPR, in order for the cells to survive, DSB has to be repaired to maintain the cellular homeostasis. The repair happens by either non-homologous end joining (NHEJ) or homology directed (HDR) means. NHEJ causes random deletion or insertion of bases. Depending on the location and accessibility of DNA, NHEJ can cause single nucleotide deletions to loss of large fragment of DNA (sometimes loss of more than half of chromosome itself is witnessed in human cells). Hence, a range of DNA fragments are generated by NHEJ from the CRISPR cleavage site and these fragments serve as templates for DNA repair elsewhere in the genome. Therefore, NHEJ based repair outcomes in the genome has the capacity to cause large scale aberrations in the genome. In contrast, HDR mediated repair utilizes endogenous DNA repair machinery to copy information from the DNA template provided externally. Thus, repair through HDR has the least chance of causing genomic aberration, hence a preferred approach for DNA repair post DSB creation. In addition, HDR is the DNA repair of choice for gene corrections (for cancer therapy and trait creation) and targeted gene insertions (for synthetic biology purposes).
However, in the somatic cells which are often the target cells used in genome editing experiments, repair mediated by NHEJ is predominant over HDR leading to low HDR efficiency in these cells. Given the importance of HDR based genome editing, there is a need to improve efficiencies in near future. There are several approaches to improve HDR efficiencies in somatic cells which include, donor template optimization, modification of donor template, modification of SSN, customizing chemicals and small molecules involved in DNA repair, as well as manipulation of cell cycle. There should be enough investment in future towards research and development to improve HDR efficiencies. Improved HDR efficiencies have capacity to create next round of revolution in genome editing which will be crucial for survival in the changing environment conditions and meet the needs of increasing human population.