BioSci D145 Lecture #8 Bruce Blumberg (blumberg@uci.edu) 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) phone 824-8573 TA – Riann Egusquiza (regusqui@uci.edu) 4351 Nat Sci 2– office hours M 1-3 Phone 824-6873 Updated lectures will be posted on web pages after lecture http://blumberg-lab.bio.uci.edu/biod145-w2017 Term papers due Friday, March 10 by 12 midnight (23:59.59) (-1 point for each day late) BioSci D145 lecture 8 page 1 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Term paper requirements and scoring Actual paper – 5 pages single spaced 1” margins (references not included). PLEASE DO NOT WRITE AN ABSTRACT Specific aims – 2 points (this should be about 3/4 to one page) Write a paragraph introducing the topic, state why it is important and what are the gaps in knowledge that you will address. State a hypothesis to be tested Enumerate 2-3 specific aims in the form of questions that test your hypothesis. Use 1-2 sentences to state how you will answer the question Finish with a paragraph describing what is innovative about your work and what impact it will have. It is VERY IMPORTANT to state the human health relevance of your research (if you are doing something biomedical) or the broader impacts on advancing the frontiers of knowledge (for something that is not relevant to human health). This is a critically important part of a grant application. You have to convince the reviewer that your work is important and worth funding. BioSci D145 lecture 1 page 2 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Term paper requirements and scoring Background and Significance – 3 points (about 1.5-2.5 pages) Briefly summarize what is known about the problem. Not a comprehensive review, just a summary of the important points. Succinctly state what is not known and why it is important that this research be done Address knowledge gaps Are you addressing something controversial? talk about the controversy and why your work will address it directly. In about one paragraph, state what is important about your proposed research and why will accomplishing it benefit the research community and world at large. i.e., what is the potential impact if you are successful Don’t repeat what was said in specific aims exactly but obviously they should be related. http://blumberg-lab.bio.uci.edu/biod145-w2017/example_grant.pdf BioSci D145 lecture 1 page 3 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Term paper requirements and scoring Research plan – 4 points (about 2.5-3 pages) In a short paragraph, state what you will do and why it is important. (I know it seems repetitive by now, but reviewers are busy and will be skimming your grant. You need to hit them over the head a few times before they will get your point). Restate each specific aim from the Specific aims section (one by one) describe what you will do to address the aim Break into subaims as appropriate State the hypothesis to be tested in each Explain the rationale Describe briefly what approach you will take Discuss what you expect to find Point out any possible problems and alternative approaches I am mostly concerned with your hypothesis and rationale here. Not an all-encompassing proposal – 4-5 years by a small team (e.g., your PhD thesis research) BioSci D145 lecture 1 page 4 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Where/when are they expressed? Known genes (e.g. from genome projects) Functional Genomics - The challenge: Many new genes of unknown function Where/when are they expressed? Known genes (e.g. from genome projects) Gene chips (Affymetrix) Microarrays (Oligo, cDNA, protein) Novel genes Differential display Expression profiling (3 papers this week) SAGE and related approaches Massively parallel sequencing Which genes regulate what other genes? ChIP (chromatin immunoprecipitation) What is the phenotype of loss-of-function? Gene targeting by homologous recombination Genome wide siRNA CRISPR-Cas9 BioSci D145 lecture 8 page 5 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Genome wide analysis of gene function Loss-of-function analysis is the most powerful way to identify gene function Direct link between genotype and phenotype Forward vs reverse genetics Forward genetics-> Reverse genetics -> Reverse genetics is much more important than forward genetics in post genomic era – why? random mutagenesis, then phenotypic analysis Identity of gene involved not known at the start associating functions with known genes Directed mutagenesis of individual genes, phenotypic analysis Because we have identified many genes by sequencing with no known functions, or even hints about function What are some useful approaches ? Mutagenesis (forward genetics) Systematically mutating each gene (required genome sequence) Random targeting with viruses or transposons, match genes later Can id new genes as well as known genes Generate phenocopies of mutant alleles RNAi (siRNA), morpholinos, virus induced gene silencing BioSci D145 lecture 8 page 6 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Construction of transgenic animals standard transgenesis Microinject DNA into a fertilized egg (mouse) or embryo (Drosophila) some embryos undergo integration of DNA into genome transgene goes to offspring in a few Why do these mice have stubby tails? Applications rescue of a mutation promoter analysis ID elements required for expression verify function of putative elements model for dominant forms of human diseases identify effects of misexpression Large scale mutagenesis Gene trap Enhancer trap RNAi BioSci D145 lecture 8 page 7 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Transgenesis is mostly a gain-of-function technique Gene targeting Transgenesis is mostly a gain-of-function technique Loss-of-function preferred for identifying gene function Targeted gene disruption is very desirable to understand function of newly identified genes e.g., from genome projects Or gene by gene produce a mutation and evaluate the requirements for your gene of interest good to create mouse models for human diseases knockout the same gene disrupted in a human and may be able to understand disease better and develop efficacious treatments excellent review is Müller (1999) Mechanisms of Development 82, 3-21. BioSci D145 lecture 8 page 8 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) enabling technology is embryonic stem (ES) cells (or iPS cells) these can be cultured but retain the ability to colonize the germ line essential for transmission of engineered mutations derived from inner cell mass of blastula stage embryos grown on lethally irradiated “feeder” cells which help to mimic the in vivo condition essential for maintaining stem cell phenotype Also problematic for human ES lines ES cells are very touchy in culture lose ability to colonize germ line with time easily infected by “mysterious microorganisms” that inhibit ability to colonize germ line ko labs maintain separate hoods and incubators for ES cell work ES cells depend critically on the culture conditions maintain an uncommitted, undifferentiated state that allows germ line transmission. BioSci D145 lecture 8 page 9 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) stopped here 2015 isolate genomic clones from ES cell library Restriction map Especially exons/introns Make targeting construct Want ~5kb genomic regions flanking targeted region Must disrupt essential exon Want no functional protein Verify in cell culture often useful to fuse reporter gene to the coding region of the protein gene expression can be readily monitored Insert dominant selectable marker within replacement region negative selection marker is located outside the region targeted to be replaced Electroporate DNA into ES cells, select colonies resistant to positive selection Integration positive cells then subjected to negative selection homologous recombinants lose this marker BioSci D145 lecture 8 page 10 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene Targeting (contd) Targeting vector Electroporate into ES cells Recombination Selection identification BioSci D145 lecture 8 page 11 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) Technique (contd) homologous recombination is verified by Southern blotting factors affecting targeting frequency (success) length of homologous regions, more is better. 0.5 kb is minimum length for shortest arm isogenic DNA (ie, from the ES cells) used for targeting construct is best locus targeted. This may result from differences in chromatin structure and accessibility Expand ES cell colonies BioSci D145 lecture 8 page 12 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) Transfer into blastocyst of recipient Implant into foster mothers (white or black) Progeny will be mixed color Breed mixed color F1 mice with homozygous white/black mice Black progeny derive from germ cells harboring the knockout Heterozygous for knockout Breed these to establish lines and determine effects of homozygous mutations BioSci D145 lecture 8 page 13 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) problems and pitfalls incomplete knockouts, ie, protein function is not lost but such weak alleles may be informative alteration of expression of adjacent genes region removed may contain regulatory elements may remove unintended genes (e.g. on opposite strand) interference from selection cassette strong promoters driving these may cause phenotypes BioSci D145 lecture 8 page 14 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Gene targeting (contd) Applications creating loss-of-function alleles introducing subtle mutations chromosome engineering marking gene with reporter, enabling whole mount detection of expression pattern (knock-in) BioSci D145 lecture 8 page 15 ©copyright Bruce Blumberg 2004-2016. All rights reserved

Example of a “knock-in” model BioSci D145 lecture 8 page 16 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Gene targeting (contd) Applications creating loss-of-function alleles introducing subtle mutations chromosome engineering marking gene with reporter, enabling whole mount detection of expression pattern (knock-in) advantages can generate a true loss-of-function alleles precise control over integration sites prescreening of ES cells for phenotypes possible can also “knock in” genes disadvantages not trivial to set up may not be possible to study dominant lethal phenotypes non-specific embryonic lethality is common (~30%) difficulties related to selection cassette BioSci 145B lecture 6 page 17 ©copyright Bruce Blumberg 2006-2016. All rights reserved

Conditional gene targeting Many gene knockouts are embryonic lethal some of these are appropriate and expected gene activity is required early others result from failure to form and/or maintain the placenta ~30% of all knockouts Clearly a big obstacle for gene analysis How can this be overcome? Generate conditional knockouts either in particular tissues or after critical developmental windows pass Sauer (1998) Methods 14, 381-392. BioSci D145 lecture 8 page 18 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Conditional gene targeting - contd Approach recombinases perform site-specific excision between recognition sites FLP system from yeast doesn’t work well Cre/lox system from bacteriophage P1 P1 is a temperate phage that hops into and out of the bacterial genome recombination requires 34 bp recognition sites locus of crossover x in P1 (loxP) Cre recombinase if loxP sites are directly repeated then deletions if inverted repeats then inversions result Chromosome deleted for target gene Target reversed e.g., Rosa26 BioSci D145 lecture 8 page 19 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Conditional gene targeting (contd) Strategy Make targeting construct (minimum needed for grant) homologous recombination, select for loss of DT-A transfect CRE, select for loss of tk Southern to select correct event Result called “floxed allele” 3 possible outcomes inject into blastocysts, select chimeras establish lines cross with Cre expressing line and analyze function X X BioSci D145 lecture 8 page 20 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Conditional gene targeting (contd) Tissue- or stage-specific knockouts from crossing floxed mouse with specific Cre-expressing line requirement for Cre lines must be well characterized promoters can’t be leaky Andras Nagy’s database of Cre lines and other knockout resources http://nagy.mshri.on.ca/cre_new/index.php BioSci D145 lecture 8 page 21 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Figure 5.25 Floxing mice pgs3e-fig-05-25-0.jpg

Conditional gene targeting (contd) advantages can target recombination to specific tissues and times can study genes that are embryonic lethal when disrupted can use for marker eviction can study the role of a single gene in many different tissues with a single mouse line can use for engineering translocations and inversions on chromosomes disadvantages not trivial to set up, more difficult than std ko but more information possible requirement for Cre lines must be well characterized regarding site and time of expression promoters can’t be leaky (expressed when/where not intended) BioSci D145 lecture 8 page 23 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Other approaches to genome manipulation Transgenic and knockout technology is species dependent (doesn’t work in all species – need ES cell equivalent). How else can we accomplish gene disruption in a targeted way ? RNAi approaches (Luo paper this week) Nuclease based methods - introduce double-stranded breaks ZFN – zinc finger nucleases TALEN nucleases Meganuclease CRISPR/Cas9 – RNA guided nuclease (Gilbert, Liu papers this week) Meganucleases Based on naturally occurring restriction enzymes with extended DNA binding specificity (relatively limited application) TALEN and ZFN nucleases Artificial fusion proteins combining an engineered DNA binding domain fused to a nonspecific nuclease domain from FokI restriction enzyme ZNF – zinc finger repeats TALE – repeats Main limitation is the sequence specificity that can be engineered in. BioSci D145 lecture 8 page 24 ©copyright Bruce Blumberg 2006-2016 All rights reserved

CRISPR-Cas gene editing CRISPR = clustered, regularly interspaced, short palindromic repeat Sander & Joung (2014) CRISPR-Cas systems for editing, regulating and targeting genomes, Nat Biotech 32: 347-355 Lander (2016) The Heroes of CRISPR Cell 164, 18-28 Cas9 – RNA-guided nuclease Functions as a bacterial immune system. Foreign DNA is incorporated between CRISPR repeat sequences, transcription generates crRNA. Hybridizes to tracrRNA (also encoded by CRISPR system), guides Cas9 nuclease to the target Cas9 nuclease introduces double stranded breaks into target Repair of this break can introduce deletions frameshifts, etc. BioSci D145 lecture 8 page 25 ©copyright Bruce Blumberg 2006-2016 All rights reserved

CRISPR-Cas gene editing CRISPR/Cas9 (contd) For gene targeting, we would fuse the target RNA to the tracrRNA and introduce this into the target cell, embryo, etc together with Cas9 nuclease Introduces ds breaks at target sequence which may introduce desirable mutations. Potential issues Can’t specify what happens after ds break (deletion, frameshift, insertion, etc Some question about specificity of the crRNA introduced – may have off-target effects Some sequence preferences of Cas9 nuclease may limit utility BioSci D145 lecture 8 page 26 ©copyright Bruce Blumberg 2006-2016 All rights reserved

CRISPR-Cas gene editing - applications BioSci D145 lecture 8 page 27 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Generating phenocopies of mutant alleles How to inactivate endogenous genes in a targeted but general way? RNAi = RNA interference Observation is that introduction of double-stranded RNAs into cells lead to destruction of corresponding mRNA (if there is one) Principle is siRNA – small interfering RNAs These generate small single stranded RNAs that target mRNAs for destruction by RISC – RNA interference silencing complex First applied in C. elegans where it works extremely well Can introduce siRNA into cells even by feeding to the worms! Works very well in Drosophila variably in mammalian cells Poorly in Xenopus – Why? Xenopus has an endogenous dsRNA helicase BioSci D145 lecture 8 page 28 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Dicer complex generates short duplexes from dsRNA in the cell RNAi (contd) Dicer complex generates short duplexes from dsRNA in the cell Important to have 2-nt overhangs siRNAs are generated from these fragments Antisense strand binds to mRNA and this recruits the RISC - RNAi silencing complex Complex leads to mRNA cleavage and destruction Two important reviews to read McManus and Sharp (2002) Nature Reviews Genetics 3, 737-747 Dykxhoorn et al. (2003) Nature Reviews, Molecular Cellular Biology 4, 457-467 BioSci D145 lecture 9 page 29 ©copyright Bruce Blumberg 2010. All rights reserved

Always form a hairpin structure with mismatches in stem RNAi (contd) Micro RNAs are small cellular RNAs that previously lacked any known function Always form a hairpin structure with mismatches in stem Micro RNAs direct gene silencing via translational repression (miRNAs) are mismatched duplexes that dicer processes into stRNAs (small temporal RNAs) Use same cellular complex as siRNAs Perfect matches -> target cleavage Imperfect matches -> translational repression of target Two important papers Giraldez et al (2005) Science 308, 833-838 (microRNAs regulate brain morphogenesis) Lecellier et al (2005) Science 308, 557-560 (microRNA mediates antiviral defenses in human cells) BioSci D145 lecture 9 page 30 ©copyright Bruce Blumberg 2010. All rights reserved

Parallels between siRNA and miRNA-directed RNAi RNAi (contd) Parallels between siRNA and miRNA-directed RNAi BioSci D145 lecture 9 page 31 ©copyright Bruce Blumberg 2010. All rights reserved

Ways to generate short RNAs that silence gene expression in vitro RNAi (contd) Ways to generate short RNAs that silence gene expression in vitro a) chemical synthesis of siRNA, introduce into cell b) synthesize long dsRNA, use dicer to chop into siRNA c) introduce perfect duplex hairpin, dicer generates siRNA d) make miRNA based hairpin, dicer generates silencing RNA Introduce into cells or organism by microinjection, transfection, etc. Expression is transient, loss of function ALWAYS partial can only generate phenotypes for a short time after introduction BioSci D145 lecture 9 page 32 ©copyright Bruce Blumberg 2010. All rights reserved

a) produce long hairpin from pol II promoter, let dicer make siRNA RNAi (contd) Ways to generate short silencing RNAs in vivo – need continuing expression to generate stable phenotype a) produce long hairpin from pol II promoter, let dicer make siRNA b) produce two transcripts from pol III promoter, let anneal in cells c) produce a short hairpin from pol III promoter (or viral vector), let dicer generate siRNAs d) produce imperfect hairpin from pol II promoter, let dicer generate miRNAs that direct gene silencing BioSci D145 lecture 8 page 33 ©copyright Bruce Blumberg 2006-2016 All rights reserved

RNAi for whole genome functional analysis RNAi (contd) RNAi for whole genome functional analysis First generate library of constructs that generates siRNA or stRNA Or synthesize a complete library of siRNAs Introduce these into cells, embyos (fly, frog, mouse) or animals (C. elegans, plants) For C. elegans, make the library in E. coli and simply feed bacteria to worms Must microinject or transfect with other animals Evaluate phenotypes IMPORTANT! RNAi is inherently transient unless you are expressing the siRNAs from a stably integrated plasmid RNAi is a knock-down (not knock-out) method. BioSci D145 lecture 9 page 34 ©copyright Bruce Blumberg 2010. All rights reserved

Phenotypic rescue by RNAi – synthetic lethal and related approaches ** How can we find other members of pathways we already know something about? Or, how can we find drugs that act on a pathway to kill cells (e.g., cancer cells?) Synthetic lethal is one relatively new and promising approach 2 mutations are synthetic lethal if either single mutation is viable but the double mutant is lethal BioSci D145 lecture 8 page 35 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Phenotypic rescue by RNAi – synthetic lethal and related approaches How can we find other members of pathways we already know something about? In cancer screening, what if we combine a mutation and a drug to find a combination that increases the kill rate, or reveals a phenotype that is similar to the total loss of function? Could find novel drug targets (pathways that kill cells in presence of siRNA+drug (usually sublethal amount of drug) Can find genes that are targeted by drugs – pathway analysis Could find new biomarkers that are required for cell viability following drug treatment BioSci D145 lecture 8 page 36 ©copyright Bruce Blumberg 2006-2016 All rights reserved

Mohr et al., 2010, Genomic Screening with RNAi: Results and Challenges. Annual Review of Biochemistry, 29: 37-64 BioSci D145 lecture 8 page 37 ©copyright Bruce Blumberg 2006-2016 All rights reserved