Genome Editing

Genome Editing Genome Editing 1234a b c d DNA structure Gene editing tools Let’s explore existing methods for gene editing... Zinc finger nucleases (ZFNs) Transcription activator-like effector nucleases (TALENs) CRISPR-Cas systems Prime editing O O O O NH NH N N N N N N N H N H N H N H H3 C NH2 NH2 H2 N Zing finger proteins (ZFPs) TALEs Fok1 nuclease domain Mechanism Why edit the genome and what are the challenges? The ability to edit the genome of living organisms presents numerous opportunities and challenges. has ushered in a “revolution” in genome-editing science since its Nobel Prize-winning discovery in 2012. Clustered Regularly Interspaced Short Palindromic Repeats Since the double DNA helix was discovered in 1953, scientists have explored different ways to edit DNA in living organisms. This process is called genome editing, and in this infographic, we’ll explore different tools that enable changes to be made to the genetic code. The ability to edit DNA requires an understanding of its molecular structure. Nucleotides are the “building blocks” of DNA. Each nucleotide is made up of a sugar molecule (deoxyribose), a phosphate group and one of four nitrogenous bases: As our understanding of DNA evolved, so has the toolbox for editing it. Now, several techniques allow scientists to manipulate the genetic code, the majority of which harness the enzymatic activity of nucleases that are programmable. The basic principle across all approaches is that the nuclease of choice is directed to a specific genome site using a molecular guide. CRISPR refers to repetitive sequences – found in bacterial genomes – that are interspaced with unique stretches of DNA that have been plucked from viruses that have previously infected the cell. Should the same virus attempt to re-invade in the future, the bacterium can produce a segment of RNA (known as a “guide RNA” or gRNA) matching the pathogenic DNA sequence that is stored in its genetic “memory book”. This RNA complex, coupled with a CRISPR-associated endonuclease (or Cas enzyme), scours the viral genome where the Cas enzyme cleaves the sequence that matches the RNA segment. This process halts viral replication, providing the bacteria with protection against infection. In 2012, scientists determined how to engineer a synthetic gRNA that guides a specific Cas enzyme – Cas9 – to any DNA sequence, unlocking a new platform for highly efficient and low-cost genome editing. Genome-editing technologies that utilize programmable nucleases result in the creation of a DSB at the target site. In the quest for an alternative method, researchers devised base editing. This technique merges a Cas9 nuclease – called a Cas9 nickase (nCas9), which selectively cleaves a single DNA strand – with enzymes capable of inducing precise conversions in DNA bases. Prime editing, also called search-and-replace genome editing, is the next evolution of base editing – the “new kid on the block”. It can be used for small insertions, deletions and base swapping. Prime editing combines nCas9 with the enzyme reverse transcriptase (RT), which generates complementary DNA from an RNA template. Collectively these components are referred to as a prime editor (PE). The prime editing guide RNA (pegRNA) is larger than the gRNAs used in other gene-editing approaches. It comprises a primer binding sequence (PBS) and the template containing the desired RNA sequence. The PE:pegRNA complex binds to the target DNA where nCas9 nicks a single strand, creating a displaced DNA loop. The PBS binds to the DNA loop and the desired RNA sequence is reverse transcribed to DNA by RT. The edited DNA is incorporated into the segment, and the target DNA is repaired with the reverse transcribed DNA. The cell’s DNA repair mechanism finalizes the editing process by excising the old sequence and sealing the new bases into the genome. The nucleotides on each strand of DNA bond with another nucleotide in a specific structure: Critical processes like DNA replication, repair and modification involve enzymes such as nucleases, helicases and polymerases that can act on DNA. Nucleases are enzymes capable of breaking the phosphodiester bonds between nucleotide bases. They can create single- or double-strand breaks (DSBs). single-strand break double-strand breaks The importance of enzymes Thymine T Cytosine C Guanine G Adenine A This is complementary base pairing. Two DNA strands wind around each other to form a DNA helix. T C G A ZFNs are artificial endonucleases engineered to consist of zinc finger proteins fused to the cleave domain of a Fok1 enzyme. TALENs are similar to ZFNs, but instead use transcription activatorlike effectors (TALEs) as DNA-binding domains. Engineered modular DNA-binding proteins that can recognize approximately three to four DNA bases. Several ZFPs can be combined to target a specific DNA sequence in the genome. TALEs were first reported in 2009, after their discovery in plantpathogenic bacteria. They are modular in structure and can be customized to recognize specific DNA sequences in the genome. ZFNs are usually designed as pairs, with one ZFN binding to each strand of the DNA target site. The Fok1 nuclease domain provides cleavage activity, creating a DSB at the target site. The binding of two TALENs at adjacent sites brings the Fok1 nuclease domains into close proximity, resulting in the creation of a DSB break which again can be repaired through NHEJ or HR. Right ZFN Right TALEN Fok1 Fok1 Fok1 Fok1 Left ZFN Left TALEN Gene Knockout Non-homologous End Joining (NHEJ) Gene Modification Homologous Recombination (HR) Guide RNA Cas9 DNA Matching genomic sequence Deleting a gene Inserting a gene Gene is disrupted Gene has a new sequence Several different types of CRISPR-Cas systems have been discovered since 2012. These systems have unique characteristics that are advantageous to different applications in genome-editing – some Cas13 enzymes can be used to edit RNA, for example. Off-target events are rare using this system. This specificity earned the TALEN system Nature Methods’ “tool of the year” title in 2011. ZFNs can be used to: • Introduce small insertions or deletions (indels) during the repair process, disrupting a gene’s function. • Insert a desired genetic modification by using a DNA template, provided by the researcher, that is incorporated during the repair process. PE has been utilized to induce specific gene modifications in a number of cell types, organoids, animal models and plants. While its precision and avoidance of DBS is advantageous, it has a lower editing efficiency compared to other techniques, which remains a barrier to its widespread use. Gene editing has the potential to reshape various facets of our lives and our planet. Its future impact will depend on how effectively we manage these wider challenges. Meanwhile, genome-editing techniques continue to grow and evolve. Recently, excitement has surged after the discovery of the first programmable RNA-guided system in eukaryotes, expanding the toolbox further still. nCas9 RT pegRNA New DNA Edited sequence Original sequence gets excised Cellular endunuclease Binding New DNA region PE: pegRNA complex RT RT Targeted medicines Biofuels Safety Diseaseresistant crops Ethical concerns New industrial products Regulatory challenges Opportunities Challenges