Introduction to CRISPR and Cas9

From UBC 2013: CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats) are specific regions in some bacterial and archaeal genomes that, together with associated Cas (CRISPR-associated) genes, function as an adaptive immune system in prokaryotes. While the specific ‘adaptive’ nature of this immunity is still under investigation, it is known that exogenous DNA is processed by Cas proteins into short (~30 base pair) sequences that are adjacent to the Protospacer Adjacent Motif (PAM) site. These short pieces of DNA are then incorporated into the host genome between repeat sequences to formspacer elements. The repeat-spacer-repeat array is constitutively expressed (pre-CRISPR RNAs or pre-crRNAs) and processed by Cas proteins to form small RNAs (crRNAs). The small RNAs are then loaded into Cas proteins and act to guide them to initiate the sequence-specific cleavage of the target sequence.

Background from Freiburg 2013: Hidden as an uncharacterized E. coli locus for more than 15 years, Barrangou et al. identified the CRISPR array as a previously unknown adaptive prokaryotic immune system. Almost half of all prokaryotes make use of this defense mechanism against unselective uptake through natural transformation, phage DNA transduction or horizontal gene transfer by conjugation. Invasive DNA or even RNA can be specifically recognized and efficiently cleaved. This unique feature results from the interaction of non-coding RNAs and CRISPR associated (Cas) proteins. From a wide range of known CRISPR subtypes we used CRISPR type II b of S. pyogenes.

The recognition and degradation of invasive DNA by CRISPR/Cas type II occurs in three steps:

  1. Acquisition: Invasive DNA is recognized via a protospacer adjacent motif (PAM) – the sequence NGG. A short sequence downstream of the PAM sequence is then integrated into the host CRISPR array and is termed spacer. Spacer sequences transcribe for CRISPR RNAs(crRNAs) which help to cleave sequence-specific invasive DNA. These sequences are located between short palindromic repeats, which are neccessary for the functionality of the crRNAs.
  2. Expression/Transcription: The Cas9 endonuclease is expressed. CRISPR array is then transcribed and processed by RNAse III into crRNAs. These contain the complementary spacer sequence and the direct repeat sequence. The crRNA guides the Cas9 protein specifically to invasive DNA sequences. Furthermore trans-activating crRNAs (tracrRNA) are transcribed and bind to the direct repeat part of the crRNA. The tracrRNA is necessary for the formation of a Cas9-RNA complex.
  3. Interference: Repeatedly invading DNA, which has been integrated into the CRISPR locus, is detected by the RNA-protein complex and cleaved by Cas9.

Each of these teams have worked on CRISPR based systems for at least some part of their projects. Below, you'll find abstracts for each team, direct links to their CRISPR pages and references. Here is the list of iGEM teams who worked on CRISPR in their projects:

Team Year Parts Track Project Title
Aachen 2015 Parts Manufacturing Upcycling Methanol into a Universal Carbon Source
BGU_Israel 2015 Parts Health & Medicine The Boomerang system: engineering logic gate genetic device for detection and treatment of cancer
BostonU 2015 Parts Foundational Advance Developing conditionally dimerizable split protein systems for genetic logic and genome editing applications
Chalmers-Gothenburg 2015 Parts New Application A study in Scarlet
Hong_Kong_HKU 2015 Parts New Application Controllable cell death and DNA degradation by CRISPR cas system
NJAU_China 2015 Parts New Application The Horcrux
Paris_Bettencourt 2015 Parts Food & Nutrition Ferment It Yourself
SCU_China 2015 Parts Environment E. pangu: The Pioneer of Mars
Stanford-Brown 2015 Parts Manufacturing biOrigami: A New Approach to Reduce the Cost of Space Missions
Tec-Monterrey 2015 Parts New Application Insects join iGEM: Sf9 cells as a new chassis for synthetic biology
Tufts 2015 Parts Health & Medicine Delivery of the CRISPR-Cas9 gene editing platform into epithelial cells using Clostridium difficile toxin B
Waterloo 2015 Parts Foundational Advance CRISPieR: re-engineering CRISPR-Cas9 with functional applications in eukaryotic systems
Yale 2015 Parts Foundational Advance Developing a Framework for the Genetic Manipulation of Non-Model and Environmentally Significant Microbes
USTC 2015 Parts Hardware NDM: Nanomachine Detecting Microbiotics
Vilnius-Lithuania 2015 Parts Foundational Advance Controlling the Lifetime of GMOs using ColiClock
Duke 2015 Parts Foundational Advance DNA Sequence Sensing with dCas9 Applied to Antibiotic Resistance Detection and Elimination
EPF_Lausanne 2015 Parts Information Processing Bio LOGIC: Biologic Orthogonal gRNA-Implemented Circuit
NU_Kazakhstan 2015 Parts Health & Medicine Prevention of Dental Caries by Targeting Streptococcus Mutans
Peking 2015 Parts Health & Medicine Fighting Against Tuberculosis: Making Invisible Visible
Tsinghua 2015 Parts Hardware Developing light-controlled systems to manipulate genetic information in prokaryotes
Washington 2015 Parts New Application Lab on a Strip: Developing a Novel Platform for Yeast Biosensors
William_and_Mary 2015 Parts Measurement Measurement of Promoter-Based Transcriptional Noise for Application in Gene Network Design
British_Columbia 2013 Parts Food & Energy ~
Chiba 2013 Parts New Application ~
Duke 2013 Parts New Application ~
Freiburg 2013 Parts Foundational Advance ~
MIT 2013 Parts Health & Medicine ~
NJU_NJUT_China 2013 Parts New Application ~
Paris_Bettencourt 2013 Parts Health & Medicine ~
Penn_State 2013 Parts Manufacturing ~
SJTU-BioX-Shanghai 2013 Parts New Application ~
Stanford-Brown 2013 Parts New Application ~
UCSF 2013 Parts Foundational Advance ~
WHU-China 2013 Parts New Application ~

CRISPR and Cas9 parts in the Registry

Many of the teams on this page have submitted parts associated with CRISPR/Cas9:

NameDescriptionLengthCreated byDocumentationUsesType
BBa_K1218003CRISPR CasA E. coli (Modern)1509Trevor Kalkus, Gordon Wade, Alissa Greenberg1523 Coding
BBa_K1218011Cas95080Sophia Liang149242Coding
BBa_K1129006Cas 9 from Streptococcus thermophilus4167UBC iGEM 201318033 Coding
BBa_K1218004CRISPR CasA (Ancestral)1340Trevor Kalkus, Gordon Wade, Alissa Greenberg1647 Coding
BBa_K1081000J13002-dcas94180Hangxing Jia3925 Composite
BBa_K1160000coding sequence of Cas9 from CRISPR system type II9159Huang Xingxu2347 Plasmid
BBa_K1150000dCas94101Freiburg 2013736742Coding
BBa_K1150017dCas9 with CMV promoter5012Freiburg 20132996 Device
BBa_K1026000Constitutively Expressed dCas9 Operon4311Hongyi WU2561 Composite
BBa_K1026002Constitutively Expressed gRNA targeting mRFP266Hongyi WU2960 Composite
BBa_K1150050Truncated CMV dCas9 Device #43626Natalie Louis and Lisa Schmunk3242 Device
BBa_K1179002Hef1A_Cas9-VP165141Brandon Nadres10280 Generator
BBa_K1137013crRNA anti KAN251Nicolas Koutsoubelis, Anne Loechner2291 Coding
BBa_K3201000nCpf1 (RNA-guided DNA nickase)3921Anastasios Galanis 2425 Coding
BBa_K1982000tCas9-CIBN (Prokaryotic LACE system)4731Zexu Li14867 Device
BBa_K1982001Prokaryotic tCAS94122Zexu Li14274 Coding
BBa_K1982006tCas9-Vp64(Prokaryotic)4368Zexu Li4537 Device
BBa_K1982002Prokaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)1854Zexu Li12563 Coding
BBa_K1982003CIBN(the N-terminal fragment of CIB1)612Zexu Li13032 Coding
BBa_K1982004tCas9-CIBN (Prokaryotic LACE system)4731Zexu Li14787 Device
BBa_K1982005CRY2-VP64(Prokaryotic LACE system)2100Zexu Li13514 Device
BBa_K201701235s + TFL consense + RSIAT-Luciferase + Tnos2836Monica Victoria Gutierrez Salazar4651 Device
BBa_K1994013sgRNA with dCas9 binding site sequence 9 and PP7 handle insert189Liam Carroll2458 RNA
BBa_K1994017sgRNA with 5' golden gate adapter and PP7 protein binding site178Liam Carroll1360 RNA
BBa_K2017000C-split Cas9 + DnaE C-intein2358Monica Victoria Gutierrez Salazar8961 Protein_Domain
BBa_K2017001N-split Cas9 + DnaE N-intein2241Monica Victoria Gutierrez Salazar9032 Protein_Domain
BBa_K201700735s:5'+ Ga20ox consense + SAGTI-Luciferase + Tnos3057Monica Victoria Gutierrez Salazar6306 Device
BBa_K201700835s + Ga20ox consense + RSIAT-Luciferase + Tnos2836Monica Victoria Gutierrez Salazar6380 Device
BBa_K201700935s + Ga20ox consense + AEK-Luciferase + Tnos2872Monica Victoria Gutierrez Salazar6116 Device
BBa_K201701135s:5' + TFL consense + SAGTI-Luciferase + Tnos3057Monica Victoria Gutierrez Salazar4724 Device
BBa_K2017010 35s + Ga20ox consense + RSIAT-TEV-Luciferase + Tnos2869Monica Victoria Gutierrez Salazar4519 Device
BBa_K2017014 35s + TFL consense + RSIAT-TEV-Luciferase + Tnos2869Monica Victoria Gutierrez Salazar4670 Device
BBa_K2017013 35s + TFL consense + AEK-Luciferase + Tnos2872Monica Victoria Gutierrez Salazar4664 Device
BBa_K1982007Eukaryotic tCAS94121Zexu Li14268 Coding
BBa_K1982008tCas9-CIBN (Eukaryotic LACE system)4731Zexu Li14761 Coding
BBa_K1982010CRY2-VP64(Eukaryotic LACE system)2100Zexu Li14604 Coding
BBa_K1982011tCas9-Vp64(Eukaryoticc)4368Zexu Li4612 Coding
BBa_K1946002sgRNA targeting LacI205Musa Efe Işılak2488 RNA
BBa_K1994021sgRNA containing two golden gate adapters157Isobel Holden3455 RNA
BBa_K1994025BsaI-GFP-dCas9 5075Egheosa Ogbomo10189 Composite
BBa_K2483005sgRNA target site couples facing each other with 6 bp spacer850Sophia Borowski3307 DNA
BBa_K2483006sgRNA target site couples facing each other with 18 bp spacer955Bryan Nowack3217 DNA
BBa_K2361000spdCas94120Mart Bartelds6415 Coding
BBa_K2361001dCas9 VRER4108Mart Bartelds6255 Coding
BBa_K2361004CRISPR array639Sebald Verkuijl1474 RNA
BBa_K2371006sgRNA generator for EML4-ALK variant A 83164Qi Xiao5823 Composite
BBa_K2371005sgRNA generator for EML4-ALK variant A 33164Qi Xiao5817 Composite
BBa_K2371004sgRNA generator for EML4-ALK variant A 23164Qi Xiao5823 Composite
BBa_K2558201dCas9 generator with Anderson weak promotor4308Tianze Huang1889 Generator
BBa_K2627004crRNA targeting GltA477Zhaoqin Zhang6629 Composite
BBa_K2660006N-Cas93459Victor Nunes de Jesus, Danielle Biscaro Pedrolli3252 Coding
BBa_K2558003dCas94107Tianze Huang81546Coding
BBa_K2627003crRNA targeting GltA162Zhaoqin Zhang6678 RNA
BBa_K2660007C-Cas9648Danielle Biscaro Pedrolli,Victor Nunes de Jesus3637 Coding
BBa_K3454000MCR-1_crRNA_A_Synthesis63Mingxuan Chi -1Other
BBa_K3454001MCR-1_crRNA_B_Synthesis63Mingxuan Chi -1Other
BBa_K2483004regulated dCas9 with sgRNAs and IAA enzymes fused to MS2 and PP710136Bryan Nowack3212 Composite
BBa_K1026001dCas94113Hongyi WU27532Coding
BBa_K1137014tracRNA-CAS9 4522Nicolas Koutsoubelis, Anne L?chner4051 Coding
BBa_K1559002rearranged CRISPR/Cas9 system without promoter5080Xiuqi (Rex) Xia24816Coding
BBa_K1559003CRISPR/Cas9 system with Anderson high-expression constitutive promoter5127Xiuqi (Rex) Xia1313 Generator
BBa_K1559004CRISPR/Cas9 system with Anderson medium-expression constitutive promoter5127Xiuqi (Rex) Xia1316 Generator
BBa_K1559005CRISPR/Cas9 system with Anderson low-expression constitutive promoter5127Xiuqi (Rex) Xia1292 Generator
BBa_K1559006CRISPR/Cas9 system with pBAD inducible promoter5222Xiuqi (Rex) Xia1291 Generator
BBa_K1559007CRISPR/Cas9 system with pLac inducible promoter5147Xiuqi (Rex) Xia1291 Generator
BBa_K1559008CRISPR/Cas9 system with pRha inducible promoter5214Xiuqi (Rex) Xia1239 Generator
BBa_K1774001Cas9 (optimized for expression in E. coli)4107HKU iGEM 201515331Coding
BBa_K1994015sgRNA with 5' golden gate adapter and COM protein binding site176Liam Carroll1355 RNA
BBa_K1994016sgRNA with 5' golden gate adapter and MS2 coat protein binding site172Liam Carroll1360 RNA
BBa_K1994019Multiple dCas9 binding site sequence239Liam Carroll13372Regulatory
BBa_K1982009Eukaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)1848Zexu Li12797 Coding
BBa_K1994044dCas9 Promoter48Egheosa Ogbomo13133Regulatory
BBa_K1982012VP64 transcription activitor246Zexu Li10998 Protein_Domain
BBa_K2483002Lac regulated dCas9 with LacI constitutively expressed5674Bryan Nowack1929 Device
BBa_K2558006gRNA targeting PhIF promotor96Tianze Huang13853RNA
BBa_K2558007gRNA targeting lux pR and RiboJ96Tianze Huang17502RNA