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Institute of Animal Technology Bulletin G CRISPR Cas9 genome editing G Branch News G AS ET Annual Lecture G Diary Dates Vol 55 No 10 October 2019

Bulletin Vol 55 No 10 October 2019 EDITOR Carole Wilson bulletineditor iat org uk ASSISTANT EDITOR Karen Dunford bulletinassistant iat org uk ADVERTISEMENT MANAGERS PRC Associates Ltd mail prcassoc co uk CONTENTS Dear Readers 5 Andrew Blake Tribute Award 6 7 Published monthly by the Institute of Animal Technology Registered Office 5 South Parade Summertown Oxford OX2 7JL IAT AS ET Charity Ball 9 CRISPR Cas9 genome editing 11 21 ISSN 0263 2861 AS ET News 22 23 Branch News 25 30 For enquiries other than Bulletin related contact IAT ADMINISTRATOR admin iat org uk or 0800 085 4380 Final copy date for December Bulletin 1st November The opinions expressed in the Bulletin do not necessarily reflect those of the Editor or the Institute AST2020 Call for Posters 26 Diary Dates 31

Bulletin October 2019 De ar Re aders Welcome to our October edition This month we are running something slightly different as you can probably tell from our front cover This month we are dedicating a large chunk of the editorial to CRISPR Cas9 genome editing Over the last few years much of our work has been transformed by so many things most importantly the raising of welfare standards and the awareness and understanding of them The science has also been changed so dramatically by the advent of genetic modification techniques firstly transgenic technologies and much more recently CRISPR Cas9 I think it is critical to understand something of the science being used and the impact this has had on our work and the animals we care for CRISPR Cas9 genome editing is both a very powerful tool and currently very widely used I owe Michelle Stewart a massive thank you for taking the time to write a refresher account explaining the concept to us all See pages 11 21 for the article Michelle s piece also gave me the opportunity to ask Tom Wheeler a very talented artist to design the front cover for us I do hope you all enjoy it There is no need to feel bereft of other more usual articles to the Bulletin we have a lot of Branch news this month How about signing up for the London quiz night Unfortunately there is no time to sign up for the West Middlesex Sundowner cruise but there is the opportunity to read all about it See pages 25 30 for it all I would also like to take this opportunity to remind everyone about the AS ET Annual Lecture this year being held at Kings College on Wednesday 16th October It will be presented by Professor Dame Frances Ashcroft and she will be discussing the molecular basis of diabetes I anticipate this to be both interesting informative and encourage everyone to buy tickets and go along see page 22 for more information Happy R eadi ng Carole 5

Bulletin October 2019 11 CRISPR Cas9 genome editing A quick refresher DNA to proteins By Michelle Stewart There are a few key concepts to remember in order to understand how CRISPR Cas9 works and why it is important Deoxyribonucleic acid DNA is the molecule that stores the information to build proteins this is not the only role of DNA as it also contains lots of other information that we are still trying to understand However for this article we Figure 1 A Double stranded DNA showing pairing between nucleotides A binds with T C binds with G Sugar in this case deoxyribose sugar is represented by the green pentagon phosphate is represented by the yellow circle B The start of a string of amino acids which will form a protein the order of the amino acids is taken from the code in the DNA ATG codes for the amino acid Methionine abbreviated to Met GTT codes for the amino acid Valine abbreviated to Val

12 October 2019 Bulletin will focus on the sections of DNA that code for proteins these are known as genes DNA is passed from one generation to the next and is often referred to as a recipe for life DNA is made of multiple copies of a unit called a nucleotide each nucleotide is made up of a phosphate a sugar and base The bases bind together to form a double helix structure with each strand bound to the opposite strand by pairing between the bases The sugar and phosphate form a backbone making up what appears to be the sides of a ladder Figure 1A The sugar and phosphate are the same throughout the DNA but there are four different types of bases called Adenine Thymine Guanine and Cytosine When the bases bind to each other forming the rungs of the ladder structure this always happens in a certain way Adenine always binds with Thymine A T and cytosine always binds with guanine C G This is called complementary base pairing and is important to remember as it is utilised in CRISPR Cas9 gene editing The order of the bases is the code to make a protein Three bases in a row codes for one amino acid There are twenty amino acids in total and by putting these amino acids together in a precise order and length a very specific protein is formed For example reading along a gene three nucleotides might be A T G this is the code for the amino acid Methionine the next three might be G T T this is the code for the amino acid Valine The cell will put these two amino acids together as the start of a protein continuing to read along the gene the rest of the amino acid sequence can be de coded and the protein synthesised Figure 1B By changing one nucleotide one letter in the code we can change the amino acid that should be in that location By removing or inserting a number of nucleotides we can change the protein in a more comprehensive way Modifying the protein allows us to study how these altered proteins affect normal cell function and allows researchers to understand how these altered or missing proteins cause disease There is a second macromolecule called RNA this is also made of nucleotides and can bind with DNA RNA is very similar to DNA the main differences being in the structure of the backbone and also that instead of the base T Thymine it has a similar base called U Uracil The sequences that bind together are complementary to each other and bind in the same way as two DNA bases C always binds with G and A always binds with U Every cell contains the complete DNA sequence for that animal Each cell contains two copies of the sequence therefore two copies of every gene Each individual version of a gene is known as an allele One allele for each gene is inherited from the mother and one allele for each gene from the father

Bulletin October 2019 13 Gene editing CRISPR Cas9 how does it work In the early 1990s researchers discovered a protein called Cas9 which is found in bacterial cells Cas9 is an endonuclease which means it is an enzyme that cuts DNA More importantly this Cas9 protein is directed to cut the DNA in a specific place using an RNA guide molecule The guide RNA is a specific sequence and binds to the complementary sequence of DNA in the genome For example if the RNA sequence is CCCGGUAA it will bind to a DNA sequence that is GGGCCATT Figure 2 The Cas9 protein and the guide fit together and scan the genome for the target When the RNA guide has bound to its complementary sequence the Cas9 protein can cut the DNA Figure 2 Cas9 protein and guide bind to the DNA at the location where the sequence in the DNA and the sequence in the guide RNA are complementary to each other C always binds with G A always binds with T or U in RNA When the guide RNA and DNA are bound the Cas9 protein cuts both strands of DNA Several years after the original discovery researchers realised that if they can modify the sequence of the RNA that guides Cas9 to a cut site they can change the place that the DNA is cut Since it is possible to synthesis RNA guides of any sequence this system can be adapted and utilised by researchers Almost 10 years ago this was proven and for the first time the CRISPR Cas9 system was used to cut DNA using an RNA guide synthesised in a laboratory Following this first experiment the system has been optimised and it is now possible to guide the Cas9 protein to any section of DNA and make a cut although there are some molecular requirements that limit this in certain locations

14 October 2019 Bulletin This is a revolutionary step because previous genome engineering methods either added DNA sequences to random places in the genome or added sequence s that were complementary to a certain section and waited for homologous recombination to occur In homologous recombination an identical or near identical complementary sequence is exchanged into the genome However homologous recombination in this context is a very rare event and has to be done in thousands of cells at once to get one that has worked accurately It is too inefficient to do this directly in mice Figure 3 The NHEJ non homologous end joining repair mechanism sticks the ends of the cut DNA back together often dropping out or adding nucleotides in the process HDR homology directed repair uses a template to copy a section of DNA into the gap made by the cut A section of template DNA can be added to the embryo at the same time as Cas9 and the guide RNA are injected In some cases the cell will use this template as the guide to fix the break By changing the sequence of the template we can specifically direct the change made to the DNA when it is repaired HDR happens less frequently than NHEJ but still frequently enough for this to be carried out in a mouse embryo

Bulletin October 2019 15 CRISPR Cas9 is a game changer because it allows cutting of the DNA almost anywhere in the genome in a very efficient way So efficient that it does not need thousands of chances in thousands of cells and can be done directly in a single cell mouse embryo Cutting the DNA is only the first step a change also needs to be made to the DNA sequence For this the cell s own repair mechanisms are utilised One of two things will happen firstly through a process called Non homologous endjoining or NHEJ the two cut ends of the DNA are forced back together Figure 3 During this process a few nucleotides are often dropped out or inserted This small insertion or deletion often referred to as an indel is usually enough to completely modify the protein Remember every three nucleotides in sequence code for one amino acid If you remove or add 1or 2 nucleotides this shifts the reading frame and changes the whole string of amino acids The second process is called Homology directed repair or HDR In this case rather than forcing the ends together the cell attempts to fix the DNA by using a template It is possible to add a synthesised template to the cell this template is made of DNA and contains sequence that is complementary to the genome sequence both sides of the cut site and also contains a change that we want to be incorporated into the DNA in the middle Figure 3 When the cell uses this template to repair the break it incorporates the required change at the same time Editing mouse genomes To introduce CRISPR Cas9 changes into mice there are six main steps In brief 1 Design how and where the change is going to be made and synthesise appropriate guide RNA template DNA and Cas9 protein reagents 2 Harvest single cell embryos from female mice these embryos are collected within 24 hours of the mice being mated 3 Using a specialised microscope and injection rig inject into each embryo the Cas9 protein and guide RNA as well as a donor template if required 4 Place the embryos back into a recipient pseudopregnant female and allow her to give birth 5 Screen the DNA of the offspring to see if they have the required DNA change

16 October 2019 Bulletin 6 Breed mice carrying the required change and thoroughly check the DNA sequence in and around the area that has been altered in their offspring This system is largely much more effective than previous methods of creating specific DNA changes Ten years ago the main method to induce a specific alteration would be to modify embryonic stem cells using homologous recombination inject the altered embryonic stem cells into mouse embryos to create chimeric mice that are a mixture of the original mouse embryo and the new embryonic stem cells then breed those mice to see if the required mutation is passed on Each step of this process is quite inefficient and often has to be repeated several times until it works When using stem cell technology it might take 3 years to get from a design to a mouse carrying the modification Now with CRISPR Cas9 the whole process can be completed in less than 6 months Although it should be noted that some types of more complex modification can take longer CRISPR Cas9 complications The CRISPR Cas9 system has greatly reduced the time to make genetically altered mouse lines and has increased the capacity to make precise mutations such as changing one specific nucleotide However it does come with some complications of which we need to be aware Mosaicism Cas9 protein guide RNA and the template DNA are injected into a rapidly developing embryo This means that cell division and duplication of the DNA is happening very quickly so quickly in fact that when the embryo divides into two cells then four cells then eight cells the elements of the CRISPR Cas9 system are often still present The Cas9 protein cuts the DNA in the single cell embryo the DNA is repaired and the cells may or may not carry the change required However Cas9 can cut again in two cells then again in four cells and again for several cell divisions Each time the DNA is repaired independently A mouse would normally carry two copies of a gene with potentially only two different versions alleles one inherited from the mother and one inherited from the father A mouse that has been injected with CRISPR Cas9 reagents may have up to 16 different versions of the modified gene as it has been modified differently in each cell during early embryo growth It seems that the Cas9 protein has usually deteriorated after the 8 cell stage therefore it is unlikely that there are more than 16 different versions of one gene Whilst 16 different versions would be a rare occurrence it is common to have 4 6 different alleles in one mouse that has undergone genome editing with CRISPR Cas9 Mice that carry differently modified versions of the same

Bulletin October 2019 17 Figure 4 A shows a single cell embryo with two versions of one gene only one strand of the DNA double helix is shown for ease Multiple copies of CRISPR Cas9 reagents are injected into this single cell embryo by microinjection B Cas9 cuts one allele and the cell repairs it in the process a cytosine C nucleotide is accidentally replaced with a guanine G The cell then divides passing on both the unmodified and modified versions of the gene to both daughter cells In each daughter cell the Cas9 protein cuts both alleles in the left cell the original C G change is present along with a deletion of one nucleotide in one allele in the second allele two nucleotides are deleted In the right daughter cell the original C G change is present along with insertion of two cytosine C nucleotides in one allele the second allele has a one nucleotide deletion These two cells will then divide again and eventually grow into a mouse that carries four different versions of this gene gene are called mosaic as different cell populations throughout the mouse will have different versions of the gene Some of the mouse tissues will have developed from cells carrying certain modifications and others developed from different cells carrying different modifications This can mean that when genotyping the alleles that are found in the ear tissue are not necessarily the alleles that are found in the sperm Mosaicism can be good and bad It means that there are more chances to cut the DNA and obtain the correct modification but it also means you could acquire the correct modification and then it gets changed in subsequent cells

18 October 2019 Bulletin The genotyping at this stage is very difficult and time consuming as the different alleles need to be disentangled These mosaic mice sometimes known as G0 for zero generation should not be used for experiments as it is impossible to know the make up of the mosaicism throughout the whole mouse G0s should be bred to wild type mice to produce G1s Each sperm or egg from the G0 carries only one copy of the genome so they can only pass on one of the alleles G1 offspring therefore will carry one allele from the G0 and one allele from the other parent usually a wild type mouse which passes on a wild type allele At this stage it is easier to genotype the mice and separate the ones that are required One G0 father may pass on 10 different alleles but each offspring will only carry one of them Figure 5 Figure 5 Cells made in Figure 4 continue to replicate to form a mouse embryo and live offspring The mouse that has grown from these cells will carry four different versions of the modified gene The different versions are not necessarily evenly spread throughout the mouse there might be some tissues that carry predominately two of the alleles as they arose from the embryonic cells that carried those two mutations After genotyping to ascertain if any of the mutations carried in this mouse are useful the G0 mouse is bred to a wild type mouse Each offspring from this mating will carry one wild type version of the gene inherited from the wildtype parent and one of the mutated copies inherited from the G0 parent At this stage the mutations are separated out and different GA lines can be established from each mouse

Bulletin October 2019 19 Breeding Establishment of new GA lines from G0 mice requires new breeding strategies as there can be many different GA lines arising from one founder with very few mice in each line Nomenclature on the cages needs to be clear as it can be very easy to mix up mice carrying similar mutations In addition the genotyping to distinguish these similar mutations can be difficult to optimise so it is possible that a mistake may not be immediately picked up by genotyping There may have to be some decisions made by the researchers to decide which alleles to follow which to archive and which to discard Not all of the changes made in the DNA will be useful Quality control Many different changes can happen to the DNA at the site where the Cas9 protein cuts Sometimes sections of DNA are cut and turned upside down sometimes a piece of DNA is cut out and inserts itself somewhere else in the genome sometimes there are duplications of sections of DNA sometimes very large deletions or insertions It is very important to carry out extensive sequencing and genotyping for a large section of DNA around the modification site to ensure the change that has happened is fully understood When using donor templates in the experiment it is also important to check by copy counting techniques that no additional copies of that template have inserted elsewhere into the genome Phenotypes in G0 mice CRISPR is so efficient that it might make mice that are homozygous for a genetic alteration i e it might alter both alleles leaving no normal version This is extremely rare with other types of genetic engineering but is much more common using CRISPR Cas9 This can lead to welfare concerns if the altered gene has detrimental effects Close welfare monitoring of all G0 mice is needed and strategies need to be in place early on to decide what to do if G0 mice become sick The ability to sperm freeze G0 males can sometimes rescue a line that would otherwise be lethal When the sperm is used to resurrect the line the IVF is done using wild type females therefore IVF offspring have only one copy of the mutated allele and do not show the detrimental effects Sometimes one mutated version is enough to cause a severe phenotype or lethality This is called haploinsufficency where one normal copy is not enough for the mouse to survive normally In these cases there is very little that can be done and it may be that other genome editing techniques need to be used to remove the gene in certain tissues only or make a different type of change Determining whether something is haploinsufficient needs careful records to be kept in the animal house as well as communication between

20 October 2019 Bulletin the animal care team and the research team to investigate which mice are affected when and how tissues are collected and thorough genotyping Unpredictability All of the complications described above lead to a certain amount of unpredictability There is a clear plan for the mutation that needs to be made however it is likely that other changes will happen in some of the embryos that are injected We do not always know the likely effect of the intended change and even less so for changes that are unintentional The level of the mosaicism in G0 mice cannot be predicted and whether the mutations will happen in both alleles forming mice with no wild type version is also difficult to foresee All this leads to additional challenges for animal care staff who need to keep meticulous records and increase welfare monitoring until the lines have been bred to G1 and onwards At this point the mutations should be characterised and there will be some experience of the welfare implications Summary CRISPR Cas9 is a mechanism discovered in bacterial cells that has been utilised to cut and modify DNA in a specific way Here we have focussed on mice but it is possible to use this system in other species this has been done successfully in rats and pigs and is being considered as a future treatment for human genetic disease although this is subject to significant ethical discussions and safety tests CRISPR Cas9 is an efficient way of modifying the genome to incorporate very specific changes and has greatly increased the capacity to create many different genetic alterations and better model aspects of human disease However it is important to carefully quality control newly generated alleles to ensure the alteration is understood and changes in breeding plans and monitoring need to be put in place to make the best use of the mice with the minimum impact on animal welfare Where did CRISPR Cas9 come from CRISPR Cas9 is part of the bacterial immune system Viruses that infect bacteria called Bacteriophages inject their own DNA or sometimes RNA into the bacteria The DNA is incorporated into the bacterial genome and the bacteria synthesises the viral proteins as if they were its own proteins The viral proteins form viral particles which build up inside the bacterial cell until it bursts open killing the cell and releasing more viruses to infect other bacteria CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats this is a way of storing DNA from previous viral infections so they can be recognised in future The bacteria use this library of viral DNA to synthesis guide RNAs which will guide the Cas9 protein to viral sequences in future infections The Cas9 snips the viral DNA sequence and the bacteria instead of

Bulletin October 2019 21 making viral proteins and bursting carries on as normal The CRISPR Cas9 system acts as a kind of immune system for the bacterial cell against the bacteriophage Figure 6 A Infection of bacteria by bacteriophage virus caused viral DNA to be incorporated into the bacterial genome The bacteria then synthesises viral proteins and bursts releasing further viruses to infect other bacteria B The CRISPR system is a way of storing viral sequences for later recognition Guide RNAs are made using previously stored viral sequence and the Cas9 protein is guided to the infected area where it cuts the viral DNA No viral proteins are made and bacteria survives

22 October 2019 Bulletin AS ET Annual Lecture London Sweetness and Light studying the molecular basis of diabetes Prof Dame Frances Ashcroft DBE BA PhD ScD FRMedSci FRS GlaxoSmithKline Royal Society Research Professor and Fellow of Trinity College University of Oxford ____________________________________________________________ 5 00pm Wednesday 16th October 2019 X V DPSXV LQJ V ROOHJH London The lecture will be followed by a wine reception ________________ Admission 10 00 Order Tickets from www as et org uk The names of the people attending each lecture will be entered in a Prize draw for a 100 Amazon Gift Voucher Sponsored by

AS ET is aBulletin charity set up to advance education and promote May 2014 excelle 15 the care Bulletin and welfare of animals used in science October 2019 23 To see how you can apply for a bursary visit our website www as et T is is a charity setset upup toto advance education and promote excellence in ET a charity advance education and promote excellence The companies listed below sponsor AS ET If you would like to join care and welfare ofof animals used in in science e care and welfare animals used science find out how to support us by looking at our website ee how you can apply forfor bursary visit our website www as et org u AS ET is ayou charity set up toa advance education and promote excellence in see how can apply a bursary visit our website www as et org set up to advance education andpromote promote excellence in AS ET charityset set up toadvance advance education excellence theAS ET care isand welfare ofup animals usededucation in science AS ET isisaaacharity charity to and excellence in in the of animals used science thecare careand andwelfare welfare ofanimals animalsused used inscience science the care and welfare of inin companies listed below sponsor AS ET If you would like to join them e companies listed If and youexcellence would excellence like join AS ETisisa acharity charitytobelow set up tosponsor advance AS ET education promote in the AS ET advance education and promote in theto care To see how you can apply for a bursary visit our website www as et org uk out how to support us by looking at our website the careto and welfare of animals used inatscience d out how support us by ourour website and welfare of animals used in looking science see howyou you canapply apply for bursary visit our www as et org uk ToTo see how can for aa bursary visit website www as et org uk To see how you can apply for a bursary visit our website www as et org uk TheSponsors companies listed below sponsor If you would like to join them To see how you can for a bursary visit website www as et org uk of AS ET areapply listed below and AS ET to find our out more please visitto the to join Thecompanies companies listed below sponsor AS ET Ifyou you join them The listed below sponsor AS ET If you would like them The companies listed below sponsor AS ET If would like to join them findwebsite out how to support us by looking at our website www as et org uk find out howto tosupport support usby by looking atour our website find how support us by looking our website find out how us looking atat Theout companies listed below sponsor AS ET If website you would like to join them find out how to support us by looking at our website

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Bulletin October 2019 27 West Middlesex Branch Sundowner Thames River Cruise I t was that time of year again time for the West Middlesex Branch to meet for the annual Thames river cruise The weather came through for us as well this year providing plenty of sunshine Some early birds met at a nearby pub for lunch and a few drinks before boarding the boat in the early evening We had a perfect summer drink of Pimm s provided by Datesand as we cruised along the Thames We were entertained throughout with a live singer guitarist and enjoyed chatting over a few more beverages with some dancing along the way Overall it was a lovely fun event attended by around 28 members West Middlesex members had reduced rate tickets supported by the branch and was a great way to see the sights that London has to offer We should thank Nilgun for coordinating such a marvellous event she even organised perfect weather Hannah Easter Branch Reporter Everyone having a good time

28 October 2019 Bulletin Still having a good time

October 2019 HMS Belfast and The Shard Bulletin 29

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Bulletin October 2019 31 DIARY Dates 10 October 11 November MRC Genetics Training Courses Details www har mrc ac uk careers andtraining training 6 November NACWO Refresher Glasgow Details info learningcurvedevelopment co uk 12 October Revised Date Inter branch Fishing Match Details wayne corylas co uk 7 November London Branch Quiz Night Details page 25 13 17 October AALAS National Meeting Denver USA Details https www aalas org nationalmeeting generalinformation registration 16 and 28 October AS ET Annual Lecture London and Manchester Details page 22 and via www as et org uk 20 October AS ET Congress Bursary Competition closes Details www as et org uk 4 5 November and 25 26 November NACWO 2 day course London and Glasgow Details info learningcurvedevelopment co uk Front cover CRISPR Cas9 by Tom Wheeler 8 November Herts and Essex Branch Halloween Quiz Details hertsessexbranch iat org uk 12 November Theatre Technician Workshop London Details info learningcurvedevelopment co uk 14 November Train the trainer in Tecniplast IVCs UK Details noleen tecniplastuk com 15 November NEE Branch Christmas Party Sno Zone Castleford Details page 30 26 28 November LASA Conference Birmingham Details http www lasa co uk meetings