Lecture 7 (Golden Gate Cloning) PDF

1 Genome editing: Generating mutants using CRISPR-Cas9 Cloning sgRNAs into plant expression vector Ø Golden Gate cloning Ø Type IIS restriction enzymes E. coli transformation ...

1 Genome editing: Generating mutants using CRISPR-Cas9 Cloning sgRNAs into plant expression vector Ø Golden Gate cloning Ø Type IIS restriction enzymes E. coli transformation  2 The most common gene cloning techniques include: A. Traditional cloning by type IIP restriction enzymes (RE) digestion & ligation B. Golden Gate assembly (or type IIS assembly), C. TA and blunt end cloning D. Recombinational cloning (Gateway) E. Gibson assembly etc. In this course, Golden Gate cloning and recombinational cloning (Gateway® technology, Invitrogen) will be used. However, we will review some of the most common cloning techniques throughout the course.  Cloning sgRNAs in plant expression vector 3 CHOPCHOP sgRNAs CBF4-sg11_For CBF4-sg1_Rev PCR + pCBC-DT1T2 (CRISPR scaffold vector) + Hot-Start Phusion Pol PCR product (insert) + pHEE401E (CRISPR plant expression vector) Golden Gate + BsaI (Type II RE) cloning + T4 DNA Ligase sg11-1 cbf4/ pHEE401E (final CRISPR expression vector)  4 Designing primers with cbf4-gRNAs Primer CBF4-sg11_For: 5’- ATATATGGTCTCGATTG-AGTTGTCCAAAGAAACGAGC-gttttagagctagaaatagcaagttaaaat -3’ Primer CBF4-sg1_Rev: 5’- ATTATTGGTCTCTAAAC-GGAGGGATCTAAAACTGCGG-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 CBF4-sg11_For BsaI BsaI GGTCTC gRNA (11) gRNA scaffold U6-26t U6-29p gRNA (1) GGTCTC CBF4-sg1_Rev Figure 1: INSERT – PCR product amplified from pCBC-DT1T2 plasmid with sg11+sg1  Overview of cloning strategy 5 INSERT PCR product amplified from BsaI gRNA BsaI gRNA (11) U6-26t U6-29p gRNA (1) pCBC-DT1T2 plasmid with scaffold sg11+sg1 Golden Gate cloning CRISPR VECTOR (pHEE401E) BsaI BsaI Contains multiple cloning site (MCS) for cloning gRNA CASSETTE gRNA ….. ….. RB U6-26p SpecR U6-26t EC1.2p Cas9 RbcS-E9t BastaR LB *KanR/SpecR in E. coli; BastaR in scaffold plants* 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 U6-26p/U6-29p RNA Polymerase III (Pol III) promoters and U6-26t Pol III terminators. Pol III promoters such as U6 are commonly used to express small RNAs. Adapted from XING et al. 2014.  PCR amplification using Hot-Start Phusion DNA Polymerase 6 Amplification of the sgRNA cassette is performed using Phusion Hot start High-Fidelity DNA Polymerase Phusion High-Fidelity DNA Polymerase was created by fusing a DNA-binding domain to a Pyrococcus-like proofreading polymerase (such as Pfu pol). Due to this unique fusion technique, Phusion DNA Polymerases have higher processivity and extremely low error rates, hence they are used for cloning and for long or difficult amplicons. Hot Start Phusion polymerase also has a reversibly bound, specific ligand, which inhibits the DNA polymerase activity at room temperature and thus prevents the amplification of nonspecific products. At polymerization temperatures, the ligand is released, rendering the polymerase fully active. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products. Error rate > 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase.  7 Golden Gate cloning with Type IIS Restriction Enzymes Type IIS restriction enzymes (RE) comprise a specific group of enzymes which recognize asymmetric DNA sequences and cleave at a defined distance outside of their recognition sequence, usually within 1 to 20 nucleotides. Type IIS RE, such as BsaI, are used for techniques such as Golden Gate cloning allowing sequence- independent cloning of genes without the need to modify them by including compatible restriction sites (scars). Advantages of cloning with Type IIS restriction enzymes: 1. Single-tube, one step cloning: digestion and ligation takes place in the same tube at the same time, 2. Scarless cloning: scar sequences are not being introduced because the overhang sequence created is not dictated by the restriction enzyme. The restriction site is eliminated from the ligated product. 3. Assembly of multiple fragments: multiple inserts can be assembled together simultaneously by using the right combination of complementary ends.  8 Type II restriction enzymes Restriction enzymes (RE) are classified according to the structure of their cleavage mode, i.e. whether the cleavage falls within the recognition site (Type IIP) or outside of the recognition site (Type IIS). Video on type II RE, from NEB https://www.neb.com/en/tools-and-resources/video-library/type-ii-restriction-enzymes ThermoFisher website with explanations https://www.thermofisher.com/ca/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction- modifying-enzymes/restriction-enzymes-thermo-scientific/fastdigest-thermo-scientific/type-iis-restriction-enzymes.html  9 Type IIP restriction enzymes Classical Type IIP RE (EcoRI, BamHI, etc.) are the best characterized and most frequently used enzymes. They recognize specific 4 to 8 nucleotide sequences that are typically palindromic They cleave within the recognition site, leaving sticky ends (5ʹ or 3ʹ overhangs) or blunt ends.  10 Type IIP restriction enzymes EcoRI Palindromic DNA recognition sequence Sticky ends after cleavage  11 Type IIS restriction enzymes Type IIS RE (BsaI, BbsI, etc.) cut DNA at a defined distance downstream of the recognition sequence (Figure 1). This is due to the enzyme architecture where the catalytic and recognition domains are separated by a polypeptide linker (Figure 2). Sequences beyond the recognition site can be ANY combination of nucleotides (N). With 256 potential overhang sequences possible, multiple fragments of DNA can be assembled using combinations of complementary overhangs. This cloning technique is widely used and is also known as Golden Gate assembly.  12 Type IIS restriction enzymes BbsI (Type IIS) asymmetric DNA sequence Figure 1. Comparison of the recognition site and cleavage site of Type II versus Type IIS restriction enzymes Figure 2. Computer generated structure of a Type IIS restriction enzyme  13 BsaI 5’ … G G T C T C (N)1 …3’ 3’ … C C A G A G (N)5 …5’ Ø In the lab, you performed Golden Gate cloning using BSaI-HFv2 (High Fidelity version 2; from NEB), which has been optimized for Golden Gate Assembly. Many Type IIS RE have been isolated from different bacteria. With different recognition sequences, activity temperature, etc. Check out the list of Type IIS RE available from NEB: https://international.neb.com/tools-and-resources/selection-charts/type-iis-restriction-enzymes  14 How does Golden Gate cloning work? The Type IIS RE, which cut the DNA downstream from their recognition sites at non-specific sites, can be used to generate DNA fragments with unique overhangs. Assembly of digested fragments then proceeds through the annealing of complementary four-base overhangs on adjacent fragments. The digested fragments and the final assembly no longer contain Type IIS recognition sites, because these recognition sites where the enzyme had bound were upstream of the cleavage, so no cutting is possible. The restriction site is eliminated from the ligation product, so digestion and ligation can be carried out simultaneously. The assembly product accumulates with time. Video on Golden Gate assembly (NEB) https://www.youtube.com/watch?v=EpHeu44hitI  15 Cloning a single fragment using Type IIS restriction enzymes The DNA fragment to be cloned can be a PCR product or cloned PCR product. Type IIS recognition sites on the fragment’s ends should be oriented such that cleavage leaves the fragment with two sticky ends but removes the enzyme binding sites (shown in purple). The recipient vector must have the two Type IIS recognition sites oriented so that cleavage leaves the linearized vector with sticky ends compatible with the insert and with the enzyme binding sites removed (Figure 3). Figure 3. Single fragment cloning  16 Cloning a multiple fragments using Type IIS restriction enzymes Each fragment has a unique set of overhangs which define the order of assembly. Each end is complementary Digestion with Type IIS RE to the end of the fragment it will be adjacent to in the final assembly. Ligation with T4 ligase The T4 ligase will join the complementary overhangs assembling the fragments into the accepting vector in the desired order (Figure 4). Figure 4. Multiple fragment cloning  17 Ligation with T4 ligase Involves the formation of phosphodiester bonds between 5'- phosphate (5’ PO4-) and 3'-hydroxyl residues (3’ OH) of the deoxyribose. This is equivalent to repairing "nicks" in duplex DNA. During in vivo DNA replication, DNA ligase catalyzes formation of 3ʹ→ 5ʹ phosphodiester bonds between the short fragments (Okazaki) of the discontinuously synthesized DNA From: Thermo Fisher strand at a replication fork. In recombinant DNA technology, purified DNA ligase is used to covalently join the ends of restriction fragments in vitro in the presence of ATP. The bacteriophage T4 DNA ligase is the preferred enzyme, because it can join both sticky and blunt- ended DNA fragments.  18 Golden Gate reaction GG reaction: Universal buffer that works for both BsaI and T4 ligase + ATP, Mg2+ (for ligase activity) BsaI RE T4 DNA ligase Ligation/digestion (cycle): à 37 ᵒC (BsaI) à 16-20 ᵒC (T4 ligase) Inactivation: à 50 ᵒC (T4) à 80 ᵒC (BsaI)  19 Golden Gate cloning: summary Golden Gate cloning utilizes Type IIS restriction enzymes, in combination with DNA ligase, in one step in a single reaction tube to insert a DNA fragment – or several DNA fragments – into a vector. The reaction is cycled repeatedly between the temperatures optimal for the restriction endonuclease (37 ᵒC) and the DNA ligase (16-23 ᵒC). ØAs a result, Golden Gate DNA assembly reduces (or eliminates) the number of steps required for cloning (Traditional cloning involves several steps) ØGolden Gate assembly can be designed to be scarless cloning (Gateway cloning produces constructs with an att recombination scar encoding eight amino acids) ØGolden Gate assembly is also less expensive than many commercial cloning methods, including Gateway®. ØIn Golden Gate assembly, however, it is necessary to ensure there is no RE cutting site within the DNA sequence to be cloned; this is not required in Gateway cloning.  20 Bacteria competent cells and transformation Next, you will prepare E. coli competent cells and transform them with the Golden Gate reaction: Here are links to videos that show procedures very similar to the one described in your manual: From NEB: https://www.neb.com/en/tools-and-resources/video-library/the-mechanism-of-transformation-with-competent-cells?autoplay=1 From ThermoFisher: https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular- biology/molecular-cloning/transformation/bacterial-transformation-workflow.html  21 Cloning and plasmid vectors Recombinant DNA technology has greatly advanced our ability to manipulate and study genes/RNA/proteins. The essence of recombinant DNA technology is the preparation of large numbers of identical DNA molecules. A DNA fragment of interest is linked through standard 3ʹ → 5ʹ phosphodiester bonds to a vector DNA molecule (recombinant DNA molecule), which can replicate when introduced into a host cell. When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector. Two types of vectors are most commonly used: E. coli plasmid vectors and bacteriophage λ vectors. From: Lodish et al. 2000  22 Plasmids Plasmids are circular, double stranded DNA molecules that can replicate in E. coli and are separate from a cell’s chromosomal DNA. E. coli plasmids have been engineered to work as cloning vectors. Plasmid vector essentials. Plasmids require: 1. A bacterial origin of replication (ori), for the propagation of the plasmid within bacteria 2. An antibiotic-resistance gene (ampr), for selection against any bacteria not carrying the plasmid. Ampr encodes the enzyme β- lactamase, which inactivates ampicillin. 3. At least one unique restriction enzyme recognition site, for the From: Lodish et al. 2000 cloning of a fragment of DNA to be studied into the plasmid, but more commonly a multiple cloning site with many restriction sites or recombination sites (eg in Gateway cloning)  23 Plasmid Elements: ori ori is a short DNA sequence (50 – 100 bases) which directs initiation of plasmid replication (by bacteria) by recruiting DNA replication machinery. The ori allows the plasmid to be copied (amplified) by bacteria, which is an important characteristic of why plasmids are convenient and easy to use. Once DNA replication is initiated at the origin (ORI), it continues in both directions around the circular molecule until the advancing growing forks merge The short segments represent the A·T and and two daughter molecules are produced. G·C base pairs connecting the complementary strands. From: Lodish et al. 2000  24 Expression Vector Expression vectors are used for gene expression (to study gene function). Aside from ori and antibiotic resistance gene, the expression vector contains also an expression cassette and selectable marker cassette for expression in different cells. In plants, these cassettes are cloned between the LB and RB of the T-DNA (see lecture 1). An expression cassette comprises three components: 1. Promoter sequence 2. DNA fragment (eg CDS/ORF) 3. Terminator of transcription (3' untranslated region that, in eukaryotes, usually contains a polyadenylation site). Different expression cassettes can be transfected into different organisms including bacteria, yeast, plants, and mammalian cells, as long as the correct regulatory sequences are used. Once transfected, the expression cassette directs the cell's machinery to make RNA and protein(s). In plants, the selectable marker can be antibiotic or herbicide resistance genes. The selectable marker cassette contains a promoter and terminator as well.  25 E. coli transformation Once a gene is cloned into a vector, the vector is then introduced into E. coli cells by transformation. E. coli transformation is a naturally occurring process in which bacterial cells take up foreign DNA at a low frequency. In molecular biology applications, bacteria are made “competent” (porous) for DNA uptake by treating bacterial cells with CaCl2 or by electroporation, by applying an electrical field. When the competent cells are mixed with the DNA from the ligation reaction and then heat-shocked at 42°C, some of the DNA is absorbed by the bacterial cells, where it begins to replicate. Transformation occurs with low frequency, and only a few cells are transformed by incorporation of a single plasmid molecule. Cells that are not transformed die on selection medium containing antibiotics. Once incorporated into a host cell, a plasmid can replicate independently of the host-cell chromosome. As a transformed cell multiplies into a colony, at least one plasmid segregates to each daughter cell. This allows to amplify the vector.  26 From: ThermoFisher Detailed description of bacterial transformation and competent cells, in ‘molecular cloning education’ at ThermoFisher website: https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation- workflow.html  27 Selection of bacteria harboring recombinant plasmid Competent cells can be transformed with a plasmid, LR reaction or ligation reaction, depending on the cloning strategy. Transformed cells are plated on selection media containing appropriate antibiotic for identification and recovery of successful transformants. If very few colonies are anticipated, the entire cell suspension may be plated. If many colonies are expected, cell Selection media (antibiotic) suspension can be diluted. Plates are incubated overnight at 37C. Successful transformants will divide and form colonies. Prolonged incubation should be avoided, as it often results in fusion of large colonies and the appearance of smaller, antibiotic-sensitive surrounding colonies (called satellite colonies) due to antibiotic breakdown around large colonies. 5-10 colonies growing on selection media are isolated and dissolved in water for colony PCR and/or overnight culture. Delivery of sgRNA in plants by Agrobacterium tumefaciens 28 and selection of cbf4 crispr mutant plants Cloning in plant expression Vector Transformation of E. coli and plasmid isolation Review Lecture 1 for Transformation of A. tumefaciens with plasmid background on Agrobacterium and T-DNA A. tumefaciens culture Selection of transformants on herbicide (Basta) Seed harvesting