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Part I: Humble beginnings Outline tracrRNA CRISPR Genomic DNA cas9 cas1 cas2 csn2 Part I: Humble beginnings Part II: Harnessing CRISPR Part III: Lessons of CRISPR 2

Part I: Humble beginnings 3

First report about transformation O’Connor C., 2008, Nature Education

First report about transformation Experiment Result S S R R S Dead S + R Something inside of S type bacteria induced “R--->S” transformation Griffith Fred, 1928, J. Hygiene

DNA could be a “Gene” Experiment Result S S S R R R R DNA Protein Lipid R Carbohydrate R Avery O. T., 1944, J. Expt. Med.

The “Gene” is DNA only 1969, Max Delbruck, Salvador E. Luria, and Alfred Hershey share the Nobel Prize in Physiology and Medicine Hershey A. D. and Chase M., 1952, J. General Physiology.

And then… Dr. Max Delbrück Dr. Alfred Hershey Dr. Salvador Luria Discoveries the genetic structure of viruses in 1952 Dr. Kary Banks Mullis Invent Polymerase chain reaction (PCR) in 1983 Dr. Frederick Sanger Contributions in DNA sequencing in 1955 Dr. James Watson and Dr. Fredrick Crick Describe structure of DNA in 1953 Dr. Arthur Kornberg Discover the enzyme DNA polymerase I in 1956 Hershey and chaseThe Nobel Prize in Physiology or Medicine 1969 "for their discoveries concerning the replication mechanism and the genetic structure of viruses“ Watson and CrickT he Nobel Prize in Physiology or Medicine 1962 ΦX174) bacteriophage was the first DNA-based genome to be sequenced. This work was completed by Fred Sanger and his team in 1977.The Nobel Prize in Chemistry 1980, "for their contributions concerning the determination of base sequences in nucleic acids" In 1956, Arthur Kornberg and colleagues discovered the enzyme DNA polymerase I, also known as Pol I, in Escherichia coli. The Nobel Prize in Physiology or Medicine 1959,  "for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid" Mullis The Nobel Prize in Chemistry 1993

Bacteriophage Humans are not alone in having to fend off pathogens; even the simplest organisms are under a constant threat of invasion. Bacteria, for example, are awash in a sea of viruses known as bacteriophages. “Every 2 days, half the bacteria on Earth are killed [by bacteriophages],” RNA sequencing was one of the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the complete genome of Bacteriophage MS2, The first full DNA genome to be sequenced was that of bacteriophage φX174 in 1977, 5386bp

short palindromic sequences

Identification of CRISPR Spacer we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action.  Clustered Regularly-Interspaced Short Palindromic Repeats CRISPR

Identification of cas genes Addison V. Wright, et al. 2016, Cell two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the wellcharacterized Cas9. we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action.  Cas, CRISPR associated proteins

CRISPR BLAST Nature rejected it In November 2003: without seeking external review; PNAS rejected it in January 2004:r lacked sufficient ‘‘novelty and importance’’ Molecular Microbiology rejected… Nucleic Acid Research rejected the paper in turn... Bolotin was the first to speculate how CRISPR conferred immunity—proposing that transcripts from the CRISPR locus worked by anti-sense RNA inhibition of phage gene expression. Although reasonable, the guess wouldprove to be wrong.

RNAi-like CRISPR-Cas system (CASS) we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas ) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. Rodolphe Barrangou and Philippe Horvath were working at Danisco, a yogurt company, and they were curious about strategies to help their yogurt cultures survive viral infections. They started infecting their bacteria with viruses and looked for viral DNA in the CRISPR spacers. Sure enough, the viral DNA was contained in the immune cells. When the DNA sequences were removed from the spacers, immunity was lost. In 2007, there was mechanistic proof that CRISPR served as a mechanism to defend against viruses using a completely novel mechanism of action.  Pourcel C, et al., Microbiology, 2005, 151(Part 3):653-663. Alexander B, et al. Microbiology, 2005, 151(Part 8):2551-2561. Makarova K S, et al. Biology Direct, 2006, 1(1):1-26.

CRISPR confers adaptive immunity By late 2004, he noticed a clear correlation between spacers and phage resistance—as would be reported just a few months later by Mojica and Vergnaud.

Programming CRISPR Complementary to: RNA and coding strand of DNA Template strand Cas3, one of the Cas proteins in E. coli K12 CRISPR/cas system Cascade, casABCDE, CRISPR-associated complex for antiviral defense Host sensitivity to phages was tested using a virulent variant of phage Lambda (lvir) First case of directly programming CRISPR-based immunity—a flu shot for bacteria. Brouns S J J, et al., Science, 2008, 321(5891):960-964.

CRISPR Targets DNA Plasmid Plasmid Self-splicing intron “From a practical standpoint, … it’s possible to manipulate genomic DNA” - Marraffini and Sontheimer Marraffini L A and Sontheimer E J. Science, 2009, 322(5909): 1843–1845.

Discovery of tracrRNA he approach yielded a striking result: the third-most abundant class of transcript—after only ribosomal RNA and transfer RNA—was a novel small RNA that was transcribed from a sequence immediately adjacent to the CRISPR locus (in the region that had caught Charpentier’s attention) and had 25 bases of near-perfect complementary to the CRISPR repeats. Model for tracrRNA-mediated crRNA maturation involving RNase III and Csn1. Black, repeat; green, spacer. tracrRNA can bind with almost perfect complementarity to each repeat sequence of the pre-crRNA. The resulting RNA duplex is recognized and site-specifically diced by RNase III in the presence of Csn1, releasing the individual repeat-spacer-repeat units (first processing event). The generated units undergo further processing within the spacer sequences resulting in mature crRNA species consisting of unique spacer-repeat sequences (second processing event) by a yet-to-be elucidated mechanism. Csn1 may also be involved in. tracrRNA was not only involved in processing crRNA but was also essential for the Cas9 nuclease complex to cleave DNAthe silencing of invading sequences. tracrRNA, trans-activating CRISPR RNA

Flexibility in CRISPR processing systems In CRISPR arrays of DNA, conserved repeats (squares) alternate with variable spacers (diamonds). During the acquisition/memory step, new spacers are incorporated as a result of invasion of the cell by a phage or plasmid. During the processing steps required to confer immunity, RNA transcripts of the CRISPR array are cleaved to give mature guide RNAs, which then target for destruction invading genomes that match the spacer. The related proteins CasE, Csy4 and Cas6 carry out the initial processing step in many organisms. Deltcheva et al.2 describe a different pathway for guide-RNA maturation that operates in Streptococcus pyogenes and certain other bacteria (boxed area). This pathway involves a trans-acting RNA (tracrRNA) that hybridizes with the spacers, leading to cleavage by RNase III. Csn1 aids in the process, and an undefined step yields mature guide RNAs. Susan G. 2011, Nature, 471(7340):588-589.

Structures of Cas proteins Csy4 bound to RNA substrate CasA The CASCADE complex Martin J, et al. 2014, Science, (6176):1215-1215; Wiedenheft B, et al., 2009, Structure, 17(6):904-912; Haurwitz R E, et al. 2010, Science, 329(5997):1355-8.

Emmanuelle Charpentier, Jennifer Doudna, Martin Jinek, Krzysztof Chylinski and Ines Fonfara

Flexibility in CRISPR processing systems

Each Cas9 nuclease domain cleaves one DNA strand Incubation of these variant Cas9 proteins with native plasmid DNA showed that dual-RNA–guided mutant Cas9 proteins yielded nicked open circular plasmids, whereas the WT Cas9 protein-tracrRNA:crRNA complex produced a linear DNA product Cas9 uses two nuclease domains to cleave the two strands in the target DNA

Cleavage sites in target DNA PAM, protospacer adjacent motif tracrRNA might be required for targetDNAbinding and/or to stimulate the nuclease activity of Cas9 downstream of target recognition. To distinguish between these possibilities, we used an electrophoretic mobility shift assay tomonitor target DNA binding by catalytically inactive Cas9 in the presence or absence of crRNA and/or tracrRNA. Minimal regions of functional tracrRNA and crRNA

Secondary structure of Dual-tracrRNA: crRNA S. pyogenes Dual-tracrRNA: crRNA guided target DNA cleavage by Cas9 is species specific L. innocua N. meningitidis Martin Jinek, etc., 2012, Science (Suppl.)

Cas9 can be programmed with sgRNA

CRISPR/Cas9 system

Crystal Structure of Cas9/gRNA Complex and Target DNA This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies. Hiroshi N., et al., Feb, 2014, Cell, 156(5):935–949

Structures of Cas proteins Upon binding the crRNA:tracrRNA guide, the two structural lobes of Cas9 reorient such that the two nucleic acid binding clefts face each other. This generates a central DNA binding channel, which allows access to dsDNA. Target DNA binding in the central channel and PAM-dependent R-loop formation result in a further structural rearrangement. Here, the nuclease domain lobe undergoes further rotation relative to the a-helical lobe, fully enclosing the DNA target, and the two nuclease domains engage both DNA strands for cleavage. Martin J, et al. Mar, 2014, Science, (6176):1215-1215;

PAM recognition regulates Cas9 nuclease activity Model for target search, recognition and cleavage by Cas9–RNA. The search initiates through random three-dimensional collisions. Cas9–RNA rapidly dissociates from non-PAM DNA, but binds PAMs for longer times and samples adjacent DNA for guide RNA complementarity, giving rise to a heterogeneous population of intermediates. At correct targets, Cas9–RNA initiates formation of an RNA–DNA heteroduplex, and R-loop expansion propagates via sequential unwinding. The DNA is cleaved, and Cas9–RNA remains bound to the cleaved products. Sternberg S H, et al. March, 2014, Nature, 507(7490):62-67.

Twenty-Year story of CRISPR Figure 2. The Twenty-Year Story of CRISPR Unfolded across Twelve Cities in Nine Countries For each ‘‘chapter’’ in the CRISPR ‘‘story,’’ the map shows the sites where the primary work occurred and the first submission dates of the papers. Green circles refer to the early discovery of the CRISPR system and its function; red to the genetic, molecular biological, and biochemical characterization; and blue to the final step of biological engineering to enable genome editing. Eric S. Lander, 2016, Cell

Part II: Harnessing CRISPR Huntington’s disease, Sickle-cell anemia, diabetes, autism, AD, cancer The cancer genome atlas, sequencing 10 million mutations related to cancer. We demonstrate CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. is possible in adult animals and has potential for correction of human genetic diseases. 32

DNA sequence disrupted NHEJ and HDR DNA DNA sequence disrupted NHEJ: Non-homologous end joining +donor DNA DNA sequence replaced HDR: homology directed repair

ZFN: Zinc finger nuclease ZFN and TALEN Jeffrey C Miller, et al. 2007, Nature Biotechnology, 25, 778 – 785. ZFN: Zinc finger nuclease Like cruise missiles attacking military targets, these proteins, called transcription activator–like effectors (TALEs), can be programmed to home in on specific DNA sequences and carry out an action once there. Each TALE repeat targets a specifi c DNA base: A, T, C, or G. The repeats are the same series of 34 animo acids (color-coded) except at positions 12 and 13, which differ depending on the base being targeted. Once he had the code in hand, Bogdanove immediately wondered if TALEs could replace zinc fi ngers as the targeting mechanism for nucleases—creating so-called TALENs. Elizabeth P. , 2012, Science TALEN: transcription activator–like effector-based nuclease

Genome Editing in Mammalian Cells I: Genome modification II: Genomic deletion Le C, F Ann R, David C, et al. 2013, Science, (6121):819-823.

Models generated by CRISPR/Cas9 system Dumpier nematodes Zebrafish embryos Fruit flies Monkey Rice Pennisi E. 2013. Science, (6148):833-6.

“Off-target" effect High efficiency (48.3%) Low efficiency (14.3%) Liang, P., et al., 2015, Protein Cell, 6(5): 363-372. (Received March 30, 2015 Accepted April 1, 2015)

Cpf1, a Single RNA-Guided Endonuclease Cpf1, CRISPR from Prevotella and Francisella 1 Various strategies have been described to reduce genome-wide off-target mutations of the commonly used SpCas9 nuclease, including: truncated sgRNAs bearing shortened regions of target site complementarity, SpCas9 mutants such as the recently described D1135E variant, paired SpCas9 nickases, and dimeric fusions of catalytically inactive SpCas9 to a non-specific FokI nuclease. Now, Zetsche et al. establish a previously uncharacterized CRISPR−Cas protein, Cpf1, as a novel tool with several advantages over Cas9. First, whereas Cas9 generates cleavage products with blunt ends, Cpf1 makes staggered cuts, resulting in a 5 ʹ overhang that improves the precision of DNA insertions. Second, unlike Cas9, Cpf1 cuts at a distal site, which preserves the seed region — essential for target recognition — for future editing. Third, the T-rich protospacer-adjustment motif (PAM; a secondary recognition site) makes Cpf1 better suited to editing AT-rich DNA than Cas9, which has a G-rich PAM. Last, Cpf1 may be easier to deliver to cells, as it is smaller and does not require a tracrRNA Cpf1 makes staggered cuts Cpf1 cuts at a distal site T-rich PAM Cpf1 may be easier to deliver to cells Zetsche B, et al., Cell, Oct. 2015, 163(3):759-771 Fagerlund R D, et al., Genome Biology, 2015, 16(1):1-3

SpCas9-HF1 The majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1 Benjamin P. Kleinsiver, et al., Nature, 2016, Received 08 November 2015, Accepted 09 December 2015

Pop in and out Two step, ‘pop in & out’ approach for the generation of CRISPR-Cas9-induced targeted mutants. a In the ‘in’ step, the targeting vector (homology regions shaded gray) introduces a tag segment that is disrupted by a loxP-flanked green fluorescent protein (GFP) reporter gene. A Cas9 and sgRNA-induced double strand break (DSB) stimulates homology-directed repair (HDR) and enables the enrichment of targeted GFP+ cells by fluorescence-activated cell sorting (FACS). In the ‘out’ step, the marker is deleted by Cre/lox-mediated recombination and GFP− cells are subsequently enriched by FACS. b Two step in & out targeting approach for the seamless removal of a marker gene. In the first step, the targeting vector introduces a nucleotide replacement (a single nucleotide polymorphism (SNP)) next to a GFP reporter. The marker is removed from the targeted allele using Cas9 and a pair of sgRNAs that recognize the end of the marker cassette. The marker gene is removed by HDR with a targeting vector to provide sequences (homology regions shaded) that are wild type except for the SNP, and GFP− cells are subsequently enriched by FACS

Utilization of CRISPR/Cas9 system Hsu, P. D., et al., 2014, Cell 157(6): 1262-1278.

Part III: Lessons of CRISPR 42

funding agencies, the general public, and aspiring researchers. The story of CRISPR is rich with lessons about the human ecosystem that produces scientific advances, with relevance to funding agencies, the general public, and aspiring researchers. The most important is that medical breakthroughs often emerge The discovery of the CRISPR loci, their biological function, and the tracrRNA all emerged not from wet-bench experiments but from open-ended bioinformatic exploration of large-scale, often public, genomic datasets. from completely unpredictable origins. Ledford H. Nature, 2015, 522(7554):20-4.

This year, several leading researchers have sounded warnings about the risks of using the CRISPR gene-editing technique to modify human and other species’ genomes in ways that could have “unpredictable effects on future generations”and “profound implications for our relationship to nature” The never-ending debates about genetically modified (GM) organisms, nuclear power, chemical toxicity and the efficacy of cancer screening should be evidence enough that science does not limit or resolve controversies about risk. Democratically weighing up the benefits and risks of gene editing and artificial intelligence is a political endeavor, not an academic one. --Daniel Sarewitz.

Collaborations on CRISPR/Cas9 system 国拜耳宣布投资3亿美金与初创基因编辑公司CRISPR Therapeutics合作开发新药用于血液疾病、失明和先天性心脏病的治疗，这也仅仅只是制药公司渴求通过CRISPR技术开发新药的最初迹象。 In the second deal of its kind, Juno Therapeutics (NASDAQ: JUNO) of Seattle has tapped CRISPR-Cas9 experts Editas Medicine, of Cambridge, MA, to help engineer the T cells of cancer patients into more efficient cancer killers. They will work together on three undisclosed research programs in both branches of Juno’s cancer immunotherapy work, known in shorthand as CAR-T and TCR therapies.  Novartis was the first to marry CRISPR-Cas9 and CAR-T the therapeutic applications of CRISPR advances in genome editing technology challenges to progressing disease models CRISPR-based genome-wide screening DNA repair the biosafety of gene editing

Acknowledgment Dr. Su Qian Dr. Erich Grimm Xiaojian Wang Kun Liu Kun, up to today, he sent me 210 emails, where 180 emails with attachment, 412 literatures in total. Acknowledgment Dr. Su Qian Dr. Erich Grimm Xiaojian Wang Kun Liu Lilly Xie