Design high specificity CRISPR Cas9 gRNAs principles and tools

thank you for joining us for this webinar on design high specificity quiz forecast my guide always principles and chores my name is Heidi Wong and I'm the service marketing manager for molecular biology services at transcript prior to joining Jen script I completed my PhD in molecular biology at Vanderbilt University working on DNA replication and the DNA repair using simian virus 40 as a model system as a basic research scientist I'm always very excited to see discoveries from basic research get transformed into innovative applications in biotechnology and medicine Krishna is so such a wonderful example Chris focused my systems are first discovered in bacteria as an adaptive immune defense mechanism that you'd utilize a short only to guide degradation of Wardian a recent development of Chris focus on I has Impala date as the most wonderful genome editing tool for diverse organisms in today's webinar I will first give you an introduction of Christic as my our start which is function as part of the bacterial adapter adaptive immune system and then review how scientists engineer the Kesner system and they exploit the host DNA repair pathways to achieve a targeted genome editing I will use mammalian system as the example throughout this webinar in the second part I will walk you through steps involved in guide on a design and provide detailed guidelines at each step to get high specificity guide our own ways that avoid off target effects I will also introduce CRISPR services and the resources offered at James page to help you achieve your research goals if you have any questions during this presentation you can stop meet them by typing them in the question views that should be shown on your screen right now I would do my best to answer some questions at the end of the presentation this webinar including the Q&A session will be archived on our website at www.ge.com slash webinars dot html' also you will receive a brief survey immediately after this webinar is over I ask that you please consider filling it out as your feedback is an invaluable resource for us to improve our services for you so what is CRISPR CRISPR stands for clustered regularly interspaced short palindromic repeats and the part of the CRISPR system is cast my guess is an acronym for CRISPR associative system which is an only guided double-stranded DNA binding protein with nucleus activity wild-type kazna is capable of generating a double-stranded cut that results in two blunt ends on the target DNA as I mentioned in the beginning CRISPR was first discovered as a part of an adaptive immune system in bacteria the type 2 CRISPR system from streptococcus pyogenes is the one that gets transformed into genome editing applications in various algorithms the type to CRISPR locals contains the coding region for the chains activating only the kes complex proteins and a region that contains spacers segregated by direct repeats when wired young ray enters the bacterial the cast complex recognized the viral DNA and the Cleaves each into small fragments which are then inserted into the CRISPR locals as spacers between repeat sequences spacers in this sense are stored as molecular signature of viral DNA once the viral DNA has been inserted into the crisper locals at spacers it now can be transcribed together with the repeats as the pre-crease for only the entrance activating crisper only hybridized to the repeat regions of the pre crista only forming an army duplex that is then processed by endogenous only three to generate the mature crisper only chains activate increased by only in the case my complex if the bacteria are exposed to the same virus again the mature crisper cast my complex they recognized the very DNA based on a DNA sequence complementarity and cleave the viral DNA by a double-stranded cut by cast 9 UK's the observation led crisper cast light system is able to make double-stranded break on target the young Lacey Qin guided by simple only DNA sequence complementarity inspired scientists to an engineered a system to perform genome editing in eukaryotes in 2013 two science papers published simultaneously from George church and the fondants lab demonstrate the ability of crispiness 9 system to perform genome editing in eukaryotic cells the essential components of the engineered CRISPR cast night system include the cast 9 Yogi's and the guide are named the guide only consists of the guide and a scaffold which is of similar function as the chains activating CRISPR only and a 20 nucleotide sequence which mimics the function of the spacer quiz focus night complex recognized the target site based on sequence complementarity with the 20 nucleotide target sequence in the guide are only the cast 9 nucleus will make a double-stranded cut only if a portal space adjacent motif or perm sequence and jiejie is prison represented at 3-prime of the target site the double-stranded cut occurs three base pairs upstream of the campsite on the target gene for adapting on Chris forecast night system in eukaryotic cells we can use either an oil one plasmid or two plasma the platform here shows the triplet plasmid strategy for mammalian cells in this platform we have one plasmid including the human colon optimize the Kesner with the with the sv40 in nuclear localization signal driven by the CMV promoter we have another plasmid encoding the guide only which contains the target size and that occurred on a scaffold driven by the u6 promoter in the oil one plasmid platform both the kesner and guide only are constructed in the same vector driven by different promoters once cassadine nucleus has generated a targeted double-stranded break on the gene of interest we can then rely on hosted young repair machinery to achieve targeted genome editing there are two pathways for the younger repel on the left is the pathway for non-homologous end joining or NH EJ which is an error-prone pathway when DNA is repaired on the base pathway the two ends simply rejoined together on random insertions or deletions also known as Intel's are introduced at the site of repair eventually leading to frameshift mutations and gene knockout on the right side is the pathway for homology directed repair or homologous recombination HR when DNA is repaired and this pathway we can utilize an exogenous longer template to achieve knocking the donor template can be either a single strand DNA or double strand DNA that has homologous arms to the regions around the double-stranded break besides the tonal template should also contain desired elements we want to introduce into the target team like a single point mutation a bunch of point mutations or you on a piece of insert cells utilizes the information on the donor template to repair the double-stranded break incorporating changes on the exogenous on the template into the endogenous copy of the target gene and the leading to precise alteration correction and the gene loci CRISPR kesner has many potential applications in research and the therapeutics because of its simplicity the hottest application is of course genome editing well Chris macchesney has the advantage of being a very simple to implement system compared to other genome editing methods such as zinc finger nucleases or talons for genome editing we can use the wild-type cast knowledge to create bubbles linear cuts deletions between two cuts or we can use the cat 9 new case which is a mutant version of kesner to create single nick or double Nick's targeted genome editing can then be achieved by exploiting DNA repair pathways as described in last slide there are also other interesting applications for Chris Burke as my by utilizing the nucleus now mutant of kes 9 which allows Castlight to become a vehicle to brain proteins or nucleic acid to target DNA regions these fusion modules can be used to regulate reorganize and visualize gene making CRISPR caste 9 and even more versatile technology though there is a lot of potential for Christakis lobbying are powerful genome editing tool there are limitations of target is a major concern the canonical guide our only target sequence is 20 nucleotide long with an GG Pam sequence adjacent at e3 prime cast I can still have nucleus activity even with one two three mismatches between the guide only and the target sites this can potentially lead to off target nucleus activity and the since cast like an induced double-stranded breaks any off target nucleus activity can cause mutations in those genes and the may even cause anchor Genesis in the next assertion I will walk you through the steps of guide on a design and recommend actions at each step to help you design high specificity guide on ways based on what we've learned about the crispiness night system our goal is to fund those accurate data on a target sites with lowest of target potential and the optimum target location to give you an overall picture here I'm showing an overview of all steps involved in guide on a design starting from target gene analysis all the way down to guide on a delivery so next let's walk through each step in great details step one target gene analysis the major purpose of this step is to make sure you get the accurate target gene sequence of your interest there are multiple ways to get the target gene sequence you can use the target gene sequence information in ncbi database or if you want to check the possibility of the invariance or if the sequence information is are known for the ship species your work with genome sequencing of the target gene is always the best way and we always recommend our clients to sequence the target gene in the cells Austrians you work ways before designing guide our name this is particularly important for knocking experiments because you will need accurate sequence information to design homologous arms for your donor template also the instruction is to be analyzed to know locations of exons or coding DNA sequence CDs and the introns such information is important for selecting optimum target location which I will cover in step 4 step two find guide only canonical sequences based on the accurate target gene sequence identified in step one we know that the canonical guide language sequence is 20 nucleotide long with an NG GPM sequence at recent to East 3 prime in mammalian system we usually use the u6 promoter to drive the expression of guide only and the G is the first nucleotide for UC u 6 promoter binding and recognition so the consensus guide only sequence sequence becomes G and 20 GT one additional note on the pam sequence so the canonical Pam sequence is ng T increased focus 9 system MHC is also considered to be a Pam but we always use ng T for better targeting efficiency in the GM sequence will be checked when evaluating of target effects after funding or to knowledge regarding a sequences in the target gene the next step is to eliminate those guide arrays with high off target potential and the return the ones with low of target effects for each colony you will need to blast the G and twenty GG sequence against the whole genome to fund the similar sequences of course the higher similarity the higher of target risk but how can we carefully Gorge the off target risk we know large twenty nucleotide plus n GG pam sequence adjacent to three prime composed the guide only target site in addition to elect according to a seed model the twenty nucleotide guide on the target site can be divided into two parts the twin and the twelve nucleotides adjacent to the pam sequence referred as the seed sequences which are most critical for binding and the cleavage by the contest line ups so any sequences in the genome that share high similarity with the seed sequences will have higher of target potential based on all those fundings we summarized three criteria for god in the off target risk first existence for pam sequences both and GG and the n AG at three prime second sequence similarity the greater the number of matches the higher risk of of target bonding third sequence similarity at three prime region increases of target risk so after this of target analysis we will probably help sell several guide only is left with low of target risk the next step step four is then to choose the guide arrays with the best locations to meet your research purpose if your goal is to knock out a gene we suggest the targeting early actions because knock out is achieved by exploiting the engage eg a repair pathway if Intel's are introduced in early Earth zones you will have a higher chance to obtain reading frameshift mutants if you want to knock out the function of a specific protein Don them Qaeda is targeting the specific donor may be selected for knocking impetus usually the target region is designated without many choices therefore we simply find data on a canonical sequences within the targeting region most likely we don't have many guidance to compel in this case we just choose the relative good ones based on off target analysis both axial and the intron can be targeted selection of exons for guiding the targeting is mostly due to the design of donor template if guide only targets an exon we when we design the donor template the guide only targeting sequence can be mutated silently with all the change in the encoded protein however if the intron is targeted mutations may be introduced into targeted sequence to avoid miss cleavage of nan a template by guiding lane so usually we mutate the pam sequence and gt2 ncg on donna template after this step you will have a few good only candidates left we recommend choosing at least two Ganga's for each target team though the efficiency of CRISPR system is very high now you have selected the best guide and a slit can potentially meet your research experiment research goals the next step is to deliver the guide arrays into cells guidance can be delivered to cells as a DNA plasmid as only or transgenic early delivery of our guide only expression plasmid or only is very efficient and can be generated a very short time frame the transgenic source of guidance of very high efficiency but it is very expensive and time consuming the most common way we use for me system.if fluid on the expression plasmid in which it contains a u6 promoter that drives the transcription of guy boundary that guy down a targeting region the guide only scaffold as well as a termination signal for guiding the transcription termination the guide only expression plasmid can be an independent one for guiding the expression owning or can be only one plasmid co expressing the Kesner nucleus so far we have gone through all the steps involved in guiding link design Y and standard of target risk is a key issue to address when designing guidance for Christmas night system several approaches have been discovered to enhance the targeting efficiency by modifying either the guide only or the Cassadine nucleus one study determined the effect of truncating the guide RNA by targeting egfp researchers were quickly able to determine large guidance sequences between 17 and the 22 base pairs in length achieved similar levels of on target gene editing at 16 base pair there was very little in their activity truncated guide always led was the linking or 18 base pairs in length were used to target three different human genes we EGF a emx one and the CL key a the 17 and 18 base pair guide RNA sequences improved specificity up to ten thousandfold so the key takeaway for from this paper is led it is beneficial to keep the targeting sequence of the guy delayed at 17 or 18 basis to reduce potential of target effects another approach is to modify the Cassadine nucleus to modify the canal nuclear systems have been developed to reduce off target effects one system is referred as the cast 90k system in this system the detail egg has nine Newton's allows for single strand nicking since cast 90 case only needs a single strand to cast my guide on a complex would be needed to generate a double-stranded break student break therefore touka donghae's would need it to be designed and the guide only would require an offset which is the distance between the two guiding ways to generate a file prime over hem using this system researchers were able to of the 1,500 fold increase in specificity compared to using a single guide angry and wild type cast 9 the other modified system is referred as the dimeric CRISPR only guided Fuqua nuclear system in this system Fuqua nucleus which is used in the challenge enum editing system was fused to a catalytically inactive kazna mutant or dead has cast 9 fuqua nucleus is introduced to increase specificity because it only has nucleus activity when is in a diner form similar as the cast 9 EK system to guide our angles would be needed to allow to cast light for quan complex to bank the target DNA and the dimerize using this system researchers fund outlet it has up to 10,000 fold less mutagenic activity compared to a single guy angry and wild type cast 9 to design guidance for the cast 9 me case or the folk one dead cast 9 you can adopt the similar principles we described for a wild-type cast 9 with attention to two special fans first you need to design to guy donghae's to target each strand of the target gene respectively and second when design paired guidance you need to consider the of talk of that which is the distance between the two galleries based on literature and our experience for the Casman ecosystem we suggest the - four to twenty base pair offset a wider range of 4-8 200 base pair is also mentioned in literature for folk 1 and death curse 9 we suggested 14 to 17 base pair offset so to make the guide only design work easier scientists at James Webb developed a leading guide on a design tool and made it freely online available to the entire research community this design tool can be accessed at James Capcom / KY dolly - design - tool dot HTML several features about this tool first of these two works for designing guidance for the wildtype cast 9 nuclear system as well as the cast 9 nickel system second it accommodates multiple target input options which means you can design kinda guidance by providing gene name in simple urging ID or gene sequences third you can use this tool to divine guidance for most popular mammalian systems including human mouse and the two cells another unique feature about this tool is that we provide off target risk scores based on sequence similarity throughout the genome risk scores range from zero the best and to 58 the worst we recommend selecting guidance with risk score less than 49 to avoid possibility of of target bonding this unique feature will enable you to easily select the guide on race with high targeting specificity besides the free guide on the online design tool we also offer to specialize the Christmas services at Jen script to meet researchers needs the the guide on reconstruct the service gives our customers the opportunity to take advantage of our best-in-class jeans interface and the molecular biology as well as our scientists expertise in designing guide erase this series covers post design and the construction it starts with the critical step of guiding lead design where our scientists were carefully designed the guide is targeting your gene of interest using our best molecular biology platform we will simplifies the guide our only targeting sequence along age into the guide on a vector and they deliver you ready to use guide array expression plasmid within 10 business days for just one night night construct which is the lowest price and available for for guidance on a design and construct service for this to is we offer a standard guide on a cloning vector for free this vector is unsuitable for use in animal malian system we can also subclone guide arrays into your own vector at an no additional cost if you choose to use our free guide on a vector we will also supply the human : optimize the caste my expression plasmid for free we keep optimizing our CRISPR platform a set of in-house developed oil 1 vectors are available now so please check back often as we will be constantly adding additional offerings to the guide and reconstructs Louise the jennchris for cell line service is a fork whisper based the genome editing so is to produce genetically modified cell line using any mammalian cell line and targeting an engine this is a comprehensive service let's start from a guide on a design to transaction and the single clone generation of a wide range of cells including difficult to transfer and the tumor cell lines we were delivered on to your target sequence validated single clone with the genome modification specified along with a detailed report on generation of the clone so all the work will be done by a dedicated group of CRISPR experts at James Beck we can also perform validation as is for you our in virtual pharmacology services of a man in validated cell function Isis including in cell westerns GPCR and ion channel asses growth and apoptosis acids and we can also provide off target analysis using finger or next-generation sequencing for deep sequencing analysis we provide both CRISPR knock out and knocking cell lines with price and at eight thousand and nine thousand dollars respectively we are running a promotion for the our cell line service in November you are eligible to receive a 30% discount of the listing price in these months so please catch this great great opportunity if you need knocking or knock out cell lines for your research to wrap up today's webinar I'd like to summarize the key points and the simplicity of CRISPR cazness system makes it an efficient and the easy to implement system for genome editing giving more researchers the opportunity to use genome editing technologies in their labs however a major concern using CRISPR Kesner is the potential for off-target effects since the case the system can tolerate one two three mismatches care focused on a design starting from target gene analysis to find canonical guide RNA sequences of target analysis location analysis and the qaida only delivery allows us to obtain triangles with lowest of target risk modified casts nine nucleases including as many case and the folk one that cast nine can improve specificity pearl guiding rays are needed for modified cast night systems chair scrapes offers a free guide on the online design tool featuring on off target risk ranking and finally we hope you will consider our jennchris for services for your research needs we offer a complete genome editing solution including guiding redesign and the construct service as well as custom cell line development so I would like to thank everyone for taking the time to join this webinar I hope this has been useful to you and let you can apply some of what I talked about today to your own Chris for genome editing projects now I will answer questions you have so the first question is can we get the slides for this presentation oh yes the recorded webinar along with the slides will be archived on our website at James Capcom slash webinars dot HTML you can download the slides approximately one week after the webinar and also you can directly email me for the slice the second question we have is do you have the nikkei's cast knife form yes so we have the nikkei's Kasner expression plasmid rhaggy a transcript and you can request for that the next question we have here is one designed on a template how long should the homologous arm be so this depends whether your donor is double stranded or single stranded don't know so for double stranded don't know we recommend 500 to 1,000 bass kale and some literature's show homologous arms around 1,000 base pair is required and single string the donor can be shorter than that and the next question is do you offer services for organisms other than mammalian cells yes the kind of reconstructed service can be offered to different organisms including mammalian bacteria a plant Joseph LA etc and our standard vectors are for mammalian cells only so you will need to supply the guidance and the Kassner expression vectors for other systems and the cell line service is for mammalian system owning and the next question is which system is better to plasmid or only one plasmid based on our experience both these films are of a similar targeting efficiency we have platforms both platforms ready at James babes and the two plasmid platform for this two plasmid platform we recommend a one two one two inspection ratio and the the next question is can I use CRISPR on primary cells yes primary cells can be done if the purpose is to obtain and knock out simple since most prior primary cells have limited lifespan so isogenic clones are difficult to obtain and the next question is are you developing online tool generating 17 to 18 base pair guide RNA sequences the answer is yes let's in our R&D developing pipeline so we are working on that and please closely watch our updates we probably will send out an email if this tool is available and the next question is do you have any experience working on plants yes we have helped the many plant biologists the design guide on ways we also synthesize the code and optimize the kesner genes for our clients so you can find some related publication in our citation database but currently we do not offer standard guide on the expression vector for plants you may need to supply the vector if you choose to use their guide only construct surveys and the next question is can i generate conditional knock out cell line using CRISPR conditional knockout cell line can be generated by combining with other elements for example the Cree loxp system however integration of loxp sites into hosted genome is a process of knocking and we have another question coming up so what should I provide if I want to use the guide on reconstruct stories from you that's very simple you just need to provide the accurate target sequence a gene sequence and specify how many guide RNA is you need we will design the Guyanese according to your specific iocation you can make an online submission of your request which is very convenient what you actually contact the tech support at gene script we provide standard expression vector for free and if you want to clone the guy down into an expression vector other than the standard vector you can just send us a small aliquots of lead vector and the next question is where copy number of target gene effect genome editing yes increasing number of gene copy reduces the chance to obtain homozygous modification of the target gene so which means that the higher the copy number of your target gene the lower chance you will obtain homozygous modifications and the next question we have is other Kesner optimized for Debra fish I believe we have done this for our clients and also you can send the encoding optimization request for us and then we can optimize the Kassner for zebrafish for you okay so I will answer one last question for today Alice is it possible to design guide on Leon generation of touching or non-coding region yeah you can find yes you can design guide I'm going to target in two regions if you can find the canonical guide on a sequence which means a pam sequence and GG exists and the guide on a targeting sequence can be identified but for the knocking experiments you will need to introduce mutations to the guide only target sequence to avoid the miss targeting of donor template by a guide on race okay so let's about war what we have for today for those unanswered questions I will post the answers to our website and together with today's webinar presentation and the Q & A and that you can also email me directly at heidi dot one at james wave comm with specific questions about this presentation so also please take a moment to complete a quick poll right after this webinar and tell us how you like today's presentation and your feedback is very helpful for us to improve our services so again thank you so much for joining us today and good luck with your future research you

Channel: GenScript USA Inc.

Views: 25,642

Rating: 4.9193549 out of 5

Keywords: CRISPR, Cas9

Id: xdv6gKe_v6A

Length: 40min 12sec (2412 seconds)

Published: Mon Nov 10 2014