Potential of Genome Editing Tools in Agriculture, Preventive Health & Diseases - Current & Future

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1 Potential of Genome Editing Tools in Agriculture, Preventive Health & Diseases - Current & Future Meng How TAN mh.tan@ntu.edu.sg or tanmh@gis.a-star.edu.sg 23 April 2018

2 The Bio-Revolution Reading and writing the genome like a hard drive =

3 The Future is Here! Actual Appearance Desired Appearance

4 The Big Question How can genetically encoded information hardwired in the cell be permanently or transiently altered? DNA Editing RNA Editing Meganucleases Zinc finger nucleases TALE nucleases A-to-I editing mediated by the ADAR enzymes C-to-U editing mediated by the APOBEC enzymes CRISPR-associated nucleases

5 Writing the genome: Homologous recombination in bacteria Homologous recombination is a process whereby nucleotide sequences are exchanged between two similar or identical molecules of DNA.

6 Bacteria vs. mammals Homologous recombination occurs at high frequency in bacteria It is widely used to inactivate ( knock out ) genes in bacteria Homologous recombination occurs at very low frequency in mammals Although it has been used to modify the mammalian genome in the past three decades, the process is slow and inefficient

7 Stimulating homologous recombination in mammals In the 1990s, scientists noticed that a double-strand break introduced artificially into the genomic target could stimulate gene targeting by homologous recombination at that locus by approximately 1000-fold. Genome engineering in mammalian cells may be improved by introducing endonucleases into the cells to generate double-strand breaks in the genomic DNA! The endonuclease has to be directed only to the correct position in the genome. Otherwise, undesired changes may be made in the cell s DNA at the wrong loci (off-target effects).

8 Early development of programmable molecular scissors Meganucleases - Found naturally in some organisms, each of which recognizes a specific 12-40bp sequence. - Genome engineering is limited by the repertoire of meganucleases available, as only a few hundreds of them are known to exist in nature and it is also difficult to alter their specificity (unprogrammable). Zinc finger nucleases and TALE nucleases - Based on the DNA-binding protein domains of naturally occurring transcription factors. - Can be programmed to target specific sequences as these protein subunits are modular and 1-to-1 rules are known. - However, they are cumbersome to assemble in practice, as multiple protein subunits have to be stitched together.

9 CRISPR-Cas9 The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a prokaryotic immune system that confers resistance to foreign genetic elements such as bacteriophages. Cas9 is an endonuclease, an enzyme specialized for cutting DNA, with two active cutting sites (HNH and RuvC), one for each strand of the double helix.

10 Gene targeting in humans using CRISPR-Cas9 Since 2013, CRISPR-Cas9 has been successfully adapted for genome engineering. CRISPR-Cas9 is popular as the targeting element is easy to design and construct

11 Different Cas9 have been deployed for genome engineering in plants and animals In general, Cas9 enzymes from different bacterial species recognize different PAMs: Cas9 from Streptococcus pyogenes: NGG Cas9 from Streptococcus thermophilus: NNAGAAW Cas9 from Staphylococcus aureus: NNGRRT Cas9 from Neisseria meningitidis: NNNNGATT Cas9 from Campylobacter jejuni: NNNNACAC

12 What happens after the double-strand break? NHEJ: Non-homologous end joining DNA repair pathway (imperfect) HDR: Homology directed repair (very accurate)

13 Timeline of CRISPR research 1987 CRISPRs were first described in E. coli as a series of short direct repeats interspaced with variable sequences. (Ishino et al., J. Bacteriol. 169: ) 2002 The term CRISPR was coined to summarize this particular family of repeats that has been found in multiple bacteria and archaea. (Jansen et al., Mol Microbiol. 43(6): ) 2005 Researchers from multiple laboratories found that CRISPR spacers matched the sequences of bacterial viruses (phages). (Bolotin et al., Microbiol. 151: ; Mojica et al., J. Mol. Evol. 60: ; Pourcel et al., Microbiol. 151: ) 2006 Hypothesis that CRISPR is an immune system in prokaryotes is proposed (Makarova et al., Biol. Direct 1:7) 2007 Researchers showed that CRISPR is a bacterial immune system. (Barrangou et al., Science 315: ) 2010 The CRISPR-Cas system was shown to cleave target plasmid or phage DNA. (Garneau et al., Nature 468: 67 71)

14 Current problems with CRISPR-Cas9 1) Efficacy the Cas9 enzyme is not reliable and does not always work 2) Specificity the Cas9 enzyme can be recruited to the wrong places in the genome, especially when there are other close matches to the spacer sequence 3) Limited targeting range the Cas9 enzyme can only cut in the presence of PAM 4) Large size Cas9 enzymes consist of more than 1000 amino acids and the large size makes in vivo delivery challenging 14

15 Examining the Cas9 crystal structure The structure of the SpCas9 complex reveals a positively charged groove that is likely to be involved in stabilizing the non-target strand of the target genomic locus. Neutralization of the positively charged residues could weaken binding, thereby requiring more stringent base pairing between the RNA guide and the target DNA strand. 15

16 Improving Cas9 specificity by limiting the amount of enzyme in the cell Rationale: There should be just enough Cas9 enzyme to cleave the intended on-target locus, but not too much Cas9 that it starts cutting off-target sites. Solution 1: Instead of transducing plasmids encoding Cas9 and a grna into the cell, one can combine purified Cas9 protein and grna oligonucleotides to form a ribonucleoprotein (RNP) complex and then introduce the RNP complex into cells instead. Solution 2: Use an inducible Cas9 system to switch on and off the editing activity as desired.

17 Some inducible Cas9 systems Split-Cas9 Intein-Cas9 ERT2-Cas9 17

18 Alternative uses of CRISPR-Cas The Cas9 enzyme has two functions, namely (1) DNA targeting and (2) DNA cleavage, that can be decoupled. When the active sites have been destroyed, the catalytically dead Cas9 (dcas9) can act as a scaffold to recruit effector domains to target genomic loci.

19 Sequence-specific genomic imaging In situ hybridization (ISH) assays are generally restricted to fixed samples because the cells need to be permeabilized and their DNA denatured before the labelled probes can bind. However, labelling of genomic loci through sequence-specific protein-dna interactions is feasible for live cell imaging. Multicolour genomic imaging can be achieved by fusing orthogonal dcas9 with different fluorescent proteins.

20 Base Editing Using Deaminases

21 Why do we want to re-write the genome? 21

22 Future therapeutics permanent cure Retinitis pigmentosa

23 Clinical trials are racing ahead Chinese researchers pushing CRISPR into therapeutic trials need to clear 1 ethical and safety review, usually at their own institutions. US researchers need to clear reviews by (1) the National Institutes of Health s (NIH s) Recombinant DNA advisory committee, (2) the Food and Drug Administration (FDA), and (3) the home institution.

24 A role for CRISPR in preventive health? The heart disease vaccine: Advances in gene editing raise the prospect of a one-off injection that could reduce the risk of cardiovascular disease. This vaccine consists of a CRISPR-Cas system that will target and inactivate the PCSK9 gene, which encodes an enzyme that raises the level of low-density lipoprotein (LDL) cholesterol in the blood. Disabling PCSK9 will therefore lower blood LDL cholesterol levels, thereby reducing the risk of heart attacks and stroke. A genome surveillance system: Cells can be engineered to constitutively express Cas9 and grnas that target harmful mutations with extraordinarily high specificity. Hence, cells that accumulate deleterious mutations will either be eliminated right away (e.g. antibiotic-resistant bacteria) or be immediately modified in such a way to minimize harm (e.g. cancer cells).

25 Genome editing in animals Deliberate In 2015, Chinese scientists used CRISPR gene editing to create customized dogs. They produced beagles with double the amount of muscle mass by deleting a gene called myostatin, which encodes a protein that inhibits muscle cell growth and differentiation. Natural The effects of losing the myostatin gene are well known from nature. One breed of ultrabeefy cattle called Belgian Blues normally lack the gene and grow to hulking size. Among dogs, the mutation occurs naturally only in one breed called whippet.

26 Pig organ transplantation Pig organs and human organs are about the same size. Scientists who dream of transplanting organs from pigs into people face a nagging question: What about PERVs? Remnants of ancient viral infections, genes from porcine endogenous retroviruses are scattered throughout the pig genome, and could infect a person who one day receives a pig s organ. US researchers used the CRISPR technology to knock out PERV genes at all 62 sites in the pig genome. They then set up a company called egenesis to focus on engineering transplantable pig organs. Genetically engineered pigs free of retroviral sequences may provide safer organs for human transplant, solving our present organ shortage problem. However, researchers will need to knock out pig genes that provoke the human immune system and insert others that will prevent toxic interactions with human blood. egenesis is currently working on such modifications.

27 CRISPR in agriculture Conventional plant breeding is unlikely to meet increasing food demands and other environmental challenges. By contrast, CRISPR technology is erasing barriers to genome editing and could revolutionize plant breeding. The common white button mushroom (Agaricus bisporus) has been modified to resist browning. The gene encoding polyphenol oxidase (PPO) an enzyme that causes browning has been knocked out using CRISPR. Fruits and vegetables that resist browning are valuable because they have a longer shelf life. Scientists have used the CRISPR-Cas9 technology to improve drought stress tolerance in rice and maize. Wheat has also been engineered to be resistant against the devastating powdery mildew fungus.

28 Controversy: Genetically modified humans? 2014 Monkeys that were genetically modified using the CRISPR-Cas system were produced Gene editing in (non-viable) human embryos was demonstrated in China, but the efficiency was low.

29 Germline genome engineering Genetic changes are passed on to future generations Problems: 1) The biggest one is that CRISPR could make off-target modifications in embryonic DNA and hence cause widespread damage to the genome. 2) On-target events can have unintended consequences. - There are limits to our understanding of human genetics, geneenvironment interactions, and disease pathways. Effects are unknown and may only emerge years after the baby is born.

30 Glimpse into the future CRISPR will enable the development of new genetic cures that were once out-of-reach. CRISPR also has the potential to address the pressing world hunger problem, especially in under developed countries. However, there is debate over whether genome engineering should ever be applied to human reproductive cells, especially since any undesirable effects can be transmitted to future generations. A regulatory framework needs to be put in place. More research is needed to resolve technical difficulties and to fully evaluate the efficacy and specificity of CRISPR-Cas technologies.

31 Ongoing CRISPR activities

32 DNA and RNA Editing (DaRE) Lab We are grateful to our funding sources: