Lecture 6 (Genome Editing by CRISPR-Cas9) Lecture Notes PDF

Genome editing by CRISPR-Cas Crystal structure of the Staphylococcus aureus 3D printed model of the Cas9 complex Cas9 complex (SaCas9) Source: Innovative Genomics Institute 1 Week...

Genome editing by CRISPR-Cas Crystal structure of the Staphylococcus aureus 3D printed model of the Cas9 complex Cas9 complex (SaCas9) Source: Innovative Genomics Institute 1 Weeks 6-12: Studying the function of the Arabidopsis CBF4 gene The Arabidopsis CBF4 gene encodes a transcription factor of the AP2/ERF family. To study its function, in the second part of BIOD21 you will: A. Generate a cbf4 knock-out mutant by genome editing with CRISPR- Cas9 technology and analyze mutant phenotype. Weeks 6-9 Design sgRNA to target CBF4 in Arabidopsis. Clone sgRNA in CRISPR expression vector by Golden Gate cloning (weeks 6-7) Transform E. coli → isolate plasmid → transform Agrobacterium tumefaciens Transform plants (Arabidopsis) → stable expression. Genotyping and phenotyping of cbf4-crispr mutants (weeks 8-9) 2 B. Generate CBF4-YFP fusion protein by Gateway recombination attL1 CBF4 attL2 X X technology to determine protein localization and overexpression 35S attR1 ccdB attR2 YFP phenotype. LR Weeks 10-12 35S attB1 CBF4 attB2 YFP Clone CBF4 into a plant expression vector containing YFP by Gateway recombination cloning Transform LR reaction into E. coli → colony PCR → plasmid isolation → sequencing (Week 10) Transform plasmid into Agrobacterium → infiltrate plant leaves (N. bentamiana) → transient expression Localize protein by confocal microscopy (Week 11) 3  The CRISPR-Cas system What is CRISPR/Cas9? CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA or trRNA) Protospacer-associated motif (PAM) CRISPR-CAS9 as tools in molecular biology Cas9 D10A variant: only nickase activity dCas9 variant: nuclease deficient Off-target Mutations Applications as a Genome-editing and Genome Targeting Tool Generating a cbf4-CRISPR vector Designing sgRNA CRISPR vector & expression cassettes Cloning strategy 4  The Nobel Prize in Chemistry 2020 The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna "for the development of a method for genome editing." Emmanuelle Jennifer Charpentier Doudna 5  What is CRISPR/Cas? The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. Figure 1 In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. From: New England Biolabs (NEB) 6  Type II CRISPR Three types of CRISPR mechanisms have been identified, of which type II CRISPR is the most studied. The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing. From: New England Biolabs (NEB) 7  Acquisition phase During the acquisition phase, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps) in the bacteria genome. See NEB web site and NEB video. From: New England Biolabs (NEB) 8  Interference phase The loci are transcribed, and transcripts are then processed to generate small RNAs (CRISPR RNA or crRNA). During the interference phase, crRNA are used to guide Cas9 endonuclease that cleaves invading DNA based on sequence complementarity to the crRNA. To recognize and cleave DNA at specific sites, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. 9  CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) Cas9’s function relies on the presence of: two nuclease domains, a RuvC- like nuclease domain and a HNH- like nuclease domain, and a PAM-interacting (PI) The Cas9-crRNA-tracrRNA complex binds to foreign DNA containing PAM. Cas9 binds and starts to domain. unwind the double strand of the foreign DNA to induce duplex formation of crRNA and foreign DNA. The HNH domain and the RuvC domain cleave the DNA strand to generate a double strand break (Ishino Y et al., 2018) 10  Protospacer-associated motif (PAM) The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, NGG, (where N can be any nucleotide) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence. Even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence. During cleavage of target DNA, the HNH and RuvC-like nuclease domains cut both DNA PAM strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. 11  CRISPR-CAS9 as tools in molecular biology The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. In 2012 the Doudna and Charpentier labs developed a simplified two-component system by combining trRNA and crRNA into a synthetic single guide RNA (sgRNA) sgRNA programmed Cas9 was shown to be as Figure 2 effective in guiding targeted gene alterations. To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. 12  The wild-type Cas9 The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the double strand break (DSB) repair machinery. DSBs can be repaired by the cellular Non- double strand break (DSB) repair machinery Homologous End Joining (NHEJ) pathway, resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made. 13 Cas9 D10A variant: only nickase activity Cong and colleagues developed a mutant form, known as Cas9 D10A, with only nickase activity (one nuclease domain is inactive). This means it cleaves only one DNA strand, and does not activate NHEJ. When provided with a homologous repair template, DNA repairs are conducted via the high- fidelity homology-directed repair (HDR) pathway only, resulting in reduced indel mutations. Figure 2 14  dCas9 variant: nuclease deficient The third variant is a nuclease-deficient Cas9 (dCas9). Mutations in the HNH and RuvC domain inactivate cleavage activity, but do not prevent DNA binding. Therefore, this variant can be used to sequence- specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool. Furthermore, it can be used as a visualization tool. For instance, dCas9 fused to GFP allows to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs. Figure 2 15  Targeting Efficiency and Off-target Mutations Targeting efficiency The Cas9 system has efficiencies up to >70% in zebrafish and plants, and 2–5% in induced pluripotent stem cells. Off-target mutations Appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region or a single base difference in the PAM sequence. Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely. Recent improvements to the CRISPR system for reducing off-target mutations have been made through modifications of the gRNA (truncated gRNA within the crRNA-derived sequence; adding two extra guanine (G) nucleotides to the 5´ end; engineering a hairpin secondary structure onto the spacer region of single guide RNAs; etc). 16 Applications as a Genome-editing and Genome Targeting Tool Since 2012, the CRISPR/Cas9 system has been successfully used to target important genes in many cell lines and organisms, including human, bacteria, zebrafish, C. elegans, plants, Xenopus tropicalis, yeast, Drosophila, monkeys, rabbits, pigs, rats and mice. Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA, it is also possible to induce large deletions (like you will do for the CBF4 gene) or genomic rearrangements, such as inversions or translocations. CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries for genomic screening. Cas9’s potential reaches beyond DNA cleavage: the dCas9 version of the CRISPR/Cas9 system can target protein domains for transcriptional regulation (activation or repression), epigenetic modification, and microscopic visualization of specific genome loci. 17  SUGGESTED READING & VIDEOS Videos on CRISPR/Cas Short CRISPR video (Nature Methods): https://www.youtube.com/watch?v=4YKFw2KZA5o Doudna “How CRISPR let us edit our DNA” (TED): https://www.ted.com/talks/jennifer_doudna_we_can_now_edit_our_dna_but_let_s_do_it_wisely Primary literature Doudna JA & Charpentier E. development of sgRNA CRISPR: Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096):816-21. doi: 10.1126/science.1225829. doi: 10.1126/science.1225829 18 Week 6-9 labs: Generating a cbf4-CRISPR vector To generate a deletion in the CBF4 gene using CRISPR-Cas9, you will need to: 1. Design single guide-RNAs (gRNA) that target CBF4 2. Clone the sgRNA in a CRISPR vector that contains the Cas9 gene and is suitable for expression in plants 3. Transform Bacteria with this vector 4. Isolate plasmid and transform Arabidopsis plants 5. Confirm deletion in CBF4 gene in cbf4-crispr mutants 19  Bioinformatics lab 4 1) Design sgRNA using Chopchop 2) Design primers containing sgRNA and restriction sites BsaI, to clone sgRNA cassette in plant expression vector pHEE401E, which harbours the Cas9 enzyme. Reference for Chopchop web tool: Labun, K., Montague, T. G., Krause, M., Torres Cleuren, Y. N., Tjeldnes, H., & Valen, E. (2019). Nucleic Acids Research. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Video from Takara: sgRNA design Video from the Innovative Genomics Institute – IGI (at UC Berkeley and UCSF): The PAM sequence 20  1. How to design sgRNA sequences CRISPR/Cas9 gene targeting requires a custom single guide RNA (sgRNA) that contains a targeting sequence (crRNA) and a Cas9 nuclease-recruiting sequence (tracrRNA, or scaffold sequence). The targeting sequence (crRNA region shown in red) is a 20-nucleotide sequence that is homologous to a region in your gene of interest and will direct Cas9 nuclease activity. 21  Guidelines to design sgRNA To select a genomic DNA region that will be targeted by CRISPR (target sequence) and design the sgRNA (corresponding to the crRNA): 1. Identify the proto-spacer adjacent motif (PAM) sequence in the gDNA. The 3' end of the gDNA target sequence must have the PAM sequence (such as 5'-NGG-3'). 2. Determine the sgRNA targeting sequence The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA). Cas9 nuclease will cleave approximately 3 bases upstream of the PAM. 3. Determine the ACTUAL sgRNA targeting sequence. The PAM sequence is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore PAM should NOT be included in the sgRNA. 4. The target sequence can be on either DNA strand Source: Takara (https://www.takarabio.com/learning-centers/gene-function/gene-editing/gene-editing-tools-and-information/how-to-design-sgrna-sequences) 22 23  Web-based tools to design sgRNAs To reduce off-target mutations, web-based tools (e.g., CRISPR Design or CHOPCHOP) were developed to: facilitate the identification of potential CRISPR target sites in the genome and assess their potential for off-target cleavage, allowing to choose the most specific crRNA for your research application. detect PAM sequences list possible crRNA sequences within a specific DNA region. In D21 labs we will use Chopchop: 24  2. Cloning gRNA: CRISPR vector & expression cassettes B gRNA U6-prom gRNA (1) U6-term scaffold C EC1.2p Cas9 RbcS-E9t Figure 1. CRISPR/Cas system. (A) The Cas9-sgRNA complex (or Cas9-crRNA-tracrRNA) binds to DNA target, and triggers double strand break (DSB) when next to a short protospacer adjacent motif (PAM, ‘NGG’ for Cas9 from S. pyogenes). (B) Expression cassette for sgRNA in plants. The U6 promoter (U6-prom) of RNA polymerase III (RNA Pol III) is used for directing high expression of sgRNA in nucleus. The U6 terminator sequence prevents run-off transcripts. Multi- sgRNA expression is achieved through multi-cassettes in one vector. Repeated elements, such as promoters, gRNA scaffold (tracrRNA) and terminators are repeated for different gRNA (crRNAs). (C) Expression cassette for Cas9 (plant codon-optimized) is controlled by the egg cell-specific promoter (EC1.2p) and terminated by the rbcS-E9 terminator. The egg cell promoter gives high efficiency of homozygous mutants in primary transgenic plants. 25  Cloning 2 gRNA in plant expression vector CHOPCHOP Since we want to delete a large region of CBF4, you will select 2 gRNAs (crRNAs), that will allow two cuts in the CBF4 gene gRNAs You will then design sgRNA primers (CBF4-sg11_For and CBF4- sg1_Rev), which include the gRNAs (crRNAs) CBF4-sg11_For CBF4-sg1_Rev Primers will be used to amplify a CRISPR cassette from the vector pCBC- + DT1T2 and include the two sgRNAs. Note: the pCBC-DT1T2 vector is pCBC-DT1T2 CRISPR vector used only to assemble a second CRISPR expression cassette PCR The PCR product will be cloned into a plant expression vector that has Cas9 and one CRISPR expression cassette (pHEE401E vector). PCR product Note: the primers will also have BsaI sites to clone the sgRNA into the + pHEE401E CRISPR CRISPR plant expression vector by Golden Gate assembly (BsaI sites plant expression vector also present in the CRISPR vector) and a sequence complementary to Golden Gate the pCBC-DT1T2 plasmid. cloning sg11-1 cbf4/ pHEE401E 26  Overview of cloning strategy 27 INSERT PCR product amplified from BsaI gRNA BsaI gRNA (11) U6-26t U6-29p gRNA (1) pCBC-DT1T2 plasmid with scaffold primers containing sg11+sg1 Golden Gate cloning CRISPR VECTOR (pHEE401E) BsaI BsaI Contains multiple cloning site (MCS) for cloning gRNA CASSETTE gRNA ….. *KanR/SpecR in E. coli; BastaR in ….. RB U6-26p SpecR U6-26t EC1.2p Cas9 RbcS-E9t BastaR LB plants* scaffold FINAL cbf4-CRISPR VECTOR (cbf4 sg11+1/pHEE401E). Contains sgRNA CASSETTES and Cas9 *KanR* in E. coli; BastaR (BsaI removed after cloning) in plants* (BsaI removed after cloning) RB gRNA gRNA Cas9 RbcS-E9t BastaR LB U6-26p gRNA(11) U6-26t U6-29p gRNA(1) U6-26t EC1.2p scaffold scaffold Expression of plant codon-optimized Cas9 is controlled by the egg cell-specific promoter (EC1.2p) and the rbcS-E9 terminator. The egg cell promoter gives high efficiency of homozygous mutants in primary transgenic plants. Expression of gRNA(11)/gRNA(1) is controlled by U6 RNA Polymerase III (Pol III) promoters and U6 Pol III terminators. Pol III promoters such as U6 are commonly used to express small RNAs. Adapted from XING et al. 2014.  Insert generated by PCR BsaI gRNA#11 U6-26t pCBC-DT1T2 vector (contains expression cassette gRNA#1 BsaI for gRNA expression) PCR Insert BsaI gRNA#11 U6-26t gRNA#1 BsaI Golden Gate Cloning pHEE401E CRISPR vector Figure 1. Schematic structure of insert generated by PCR, using pCBC-DT1T2 vector as template and primers containing two sgRNAs and the BSAI sites (Week 5 lab). FOR primer anneals to the vector sgRNA scaffold sequence (Sc) and contains gRNA#11 + BsaI REV primer anneals to vector U6-29p sequence and contains gRNA#1 + BsaI. Template: pCBC-DT1T2 vector contains expression cassette for gRNA expression PCR amplicon (Insert) will be ligated into pHEE401E CRISPR vector by Golden Gate cloning using BsaI. 28  Primers with cbf4-gRNAs Primer CBF4-sg11_For: 5’- ATATATGGTCTCGATTG-AGTTGTCCAAAGAAACGAGC-gttttagagctagaaatagcaagttaaaat -3’ Primer CBF4-sg1_Rev: 5’- ATTATTGGTCTCTAAAC-CCGCAGTTTTAGATCCCTCC-caatctcttagtcgactctaccaata -3’ overhang with BsaI sites sgRNA sequence; does primer sequence that is (BsaI sites also present in NOT include the PAM complementary to the pCBC- the CRISPR vector) sequence DT1T2 vector template 29  Final CRISPR plant expression vector After the PCR product is cloned into a CRISPR plant expression vector, you will generate the final cbf4-CRISPR vector. Expression of gRNA(11)/gRNA(1) is controlled by two U6 RNA Polymerase III (Pol III) promoters and U6 Pol III terminators. Expression of plant codon-optimized Cas9 is controlled by the egg cell-specific promoter (EC1.2p) and the rbcS-E9 terminator. The egg cell promoter gives high efficiency of homozygous mutants in primary transgenic plants. The final cbf4-CRISPR vector is NOT resistant to Spec, only Kan (why?). PCR product Promoter 1 Promoter 2 Promoter 3 Terminator 1 Terminator 2 Terminator 3 RB gRNA gRNA Cas9 RbcS-E9t BastaR LB U6-26p gRNA(11) U6-26t U6-29p gRNA(1) U6-26t EC1.2p scaffold scaffold gRNA(11) gRNA(1) Cas9 expression cassette expression cassette expression cassette KanR FINAL cbf4-CRISPR VECTOR (cbf4 sg11+1/pHEE401E). BastaR expression cassette KanR expression cassette Promoter + BAR gene + terminator Contains 2 sgRNA cassettes and Cas9 Promoter + NPTII gene + terminator 30 Questions?