Essentials of Genetics, Recombinant DNA Technology PDF

11/6/2024 Essentials of Genetics Tenth Edition, Global Edition Chapter 17 Recombinant DNA Technology...

11/6/2024 Essentials of Genetics Tenth Edition, Global Edition Chapter 17 Recombinant DNA Technology Copyright © 2021 Pearson Education Ltd. All Rights Reserved Chapter Contents 17.1 Recombinant DNATechnology Began with Two Key Tools: Restriction Enzymes and Cloning Vectors 17.2 DNA Libraries Are Collections of Cloned Sequences 17.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA 17.4 Molecular Techniques for Analyzing DNA and RNA 17.5 DNA Sequencing Is the Ultimate Way to Characterize DNA at the Molecular Level 17.6 Creating Knockout and Transgenic Organisms for Studying Gene Function 17.7 Genome Editing with CRISPR-Cas Copyright © 2021 Pearson Education Ltd. All Rights Reserved 1  11/6/2024 Introduction Recombinant DNA Technology: – Also called gene splicing – DNA created by joining together pieces of DNA from various sources (recombinant DNA) – Allows for isolation and study of specific DNA sequences. Clones: recovered copies of recombinant DNA molecule: – Used to study structure and orientation of DNA Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and Cloning Vectors Copyright © 2021 Pearson Education Ltd. All Rights Reserved 2  11/6/2024 Tools of Recombinant DNA Technology Two important tools used to construct and amplify DNA molecules: – Restriction enzymes: ▪ DNA-cutting enzymes – Cloning vectors Copyright © 2021 Pearson Education Ltd. All Rights Reserved Restriction Enzymes Restriction enzymes: – Produced by bacteria as defense mechanism against infection by bacteriophage – Restrict or prevent infection by degrading DNA of invading virus. – Bind to DNA at specific recognition sequence (restriction site) and cuts DNA to produce restriction fragments (Figure 17-1). – Enzyme cleaves both strands of DNA (digestion). Copyright © 2021 Pearson Education Ltd. All Rights Reserved 3  11/6/2024 Figure 17-1 Common restriction enzymes, with their recognition sequence, DNA cutting patterns, and source microbes. Arrows indicate the location in the DNA cut by each enzyme. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Palindromes Palindrome: (‫)متناضر‬ – Symmetry exhibited by recognition sequences: ▪ Nucleotide sequence reads the same on both strands. – Restriction enzymes cut DNA in characteristic cleavage pattern (Figure 17-1). – Sticky ends (cohesive ends): fragments produced with overhangs – Blunt ends: fragments produced with double- stranded ends Copyright © 2021 Pearson Education Ltd. All Rights Reserved 4  11/6/2024 EcoRI Restriction enzyme identified in Escherichia coli: – Produces DNA fragments with cohesive ends. – ssDNA fragments from different sources can anneal (stick together) by hydrogen bonding. DNA ligase: – DNA fragments will seal phosphodiester backbone. – Joins restriction fragments covalently to produce intact DNA molecules. – Figure 17-2 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-2 DNA from different sources is cleaved with EcoRI and mixed to allow annealing. The enzyme DNA ligase forms phosphodiester bonds between these fragments to create a recombinant DNA molecule. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 5  11/6/2024 Play: Recombinant DNA Technology: Restriction Enzymes Copyright © 2021 Pearson Education Ltd. All Rights Reserved Cloning Vectors Cloning Vectors—DNA molecules that accept DNA fragments: – Can replicate cloned DNA fragments in host cell. – Must be able to replicate independent of host chromosome(s). – Have several restriction enzyme sites to allow insertion of DNA fragment. – Carry selectable gene marker/reporter gene to distinguish host cells that have taken them up from those that have not. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 6  11/6/2024 Bacterial Plasmid Vectors Plasmids used in DNA cloning: – Genetically modified bacterial plasmids—first vectors developed – Engineered to contain: ▪ Multiple cloning sites: – Short sequence with restriction sites for common restriction enzymes ▪ Figure 17.3 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-3 (a) Color-enhanced electron micrograph of plasmids isolated from E. coli. (b) Diagram of a typical DNA cloning plasmid. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 7  11/6/2024 Play: Recombinant DNA Technology: Vectors Copyright © 2021 Pearson Education Ltd. All Rights Reserved Transformation (1 of 2) Plasmids are introduced into bacteria via transformation: Two main techniques: 1. Using calcium ions and brief heat shock to pulse DNA into cells 2. Electroporation: a brief but high-intensity pulse of electricity to move DNA into bacterial cells. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 8  11/6/2024 Transformation (2 of 2) Cloning DNA with plasmid vector: – Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme. – DNA restriction fragments from DNA to be cloned are added to linearized vector in presence of DNA ligase. – Sticky ends anneal. Recombinant DNA is produced and introduced into bacterial host cells by transformation. Figure 17-4 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-4 Cloning with a plasmid vector. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 9  11/6/2024 Blue-White Screening Not all plasmids will incorporate DNA to be cloned: – Need to identify which ones are recombinant. Selectable marker genes: – Genes provide resistance to antibiotics (ampR ). Blue-white selection: – Used to identify cells containing recombinant and nonrecombinant DNA – Plasmid contains lacZ gene, which encodes β-galactosidase. – Figure 17-5 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Blue-White Screening Mechanism Blue-white screening mechanism: – Agar plates contain X-gal: ▪ Analog of lactose—substrate for β-galactosidase. – When X-gal is cleaved by enzyme, it turns blue. Bacterial cells with functional lacZ gene carrying a nonrecombinant plasmid = blue. Bacterial cells with recombinant plasmid = white. Figure 17-5 Copyright © 2021 Pearson Education Ltd. All Rights Reserved 10  11/6/2024 Figure 17-5 In blue-white screening, DNA inserted into the multiple cloning site of a plasmid disrupts the lacZ gene. Bacteria containing recombinant DNA are unable to metabolize X-gal, resulting in white colonies and allowing direct identification of colonies that carry DNA inserts to be cloned. (Bottom) Photo of a Petri dish showing the growth of bacterial cells after uptake of plasmids. Cells in blue colonies contain vectors without DNA inserts (nonrecombinant plasmids), whereas cells in white colonies contain vectors carrying DNA inserts (recombinant plasmids). Nontransformed cells did not grow into colonies due to the presence of ampicillin in the plating medium. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Other Types of Cloning Vectors Phage vectors: – Among earliest vectors used in addition to plasmids – Include genetically modified strains of bacteriophage (λ). – Have multiple cloning site. – Carry up to 45 kb of cloned DNA. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 11  11/6/2024 Larger Cloning Vectors Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs): – Vectors used to clone large fragments of DNA – BACs: ▪ Generally very large but low copy number (one to two copies/bacterial cell) plasmids ▪ DNA inserts 100–300 kb range. – YACs: ▪ Have telomeres at each end, origin of replication (ori), and centromere. ▪ Up to 1000 kb of DNA insert possible Copyright © 2021 Pearson Education Ltd. All Rights Reserved Expression Vectors Expression vectors: – Designed to ensure mRNA expression of cloned gene—to produce many copies of encoded protein in host cell – Plasmids, phage vectors, and YACs only carry DNA and do not signal for protein. Expression vectors available for both prokaryotic and eukaryotic host cells: – Ti plasmid and soil bacterium can be used for introducing genes in plants. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 12  11/6/2024 17.2 DNA Libraries Are Collections of Cloned Sequences Copyright © 2021 Pearson Education Ltd. All Rights Reserved Genomic Libraries Genomic library: – Contains of many overlapping fragments of the genome. – Has at least one copy of every DNA sequence in organism’s chromosomes. – Constructed by cutting genomic DNA with restriction enzymes and ligating fragments into vectors. – Libraries built from eukaryotic cells will contain coding and noncoding segments such as introns. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 13  11/6/2024 cDNA Library (1 of 2) Complementary DNA (cDNA) library: – Contains complementary DNA copies (cDNA) made from mRNAs isolated from cultured cells or tissues. – Represents only genes that were expressed at the time library was made: ▪ Useful for identifying genes involved in cancer formation – Genomic libraries contain all of the DNA. Copyright © 2021 Pearson Education Ltd. All Rights Reserved cDNA Library (2 of 2) cDNA library is constructed by: – Isolating mRNA from cells. – Synthesizing complementary DNA using reverse transcriptases. – Cloning cDNA molecules into vector. Figure 17-6 Copyright © 2021 Pearson Education Ltd. All Rights Reserved 14  11/6/2024 Figure 17-6 Producing cDNA from mRNA. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Reverse Transcriptase Reverse transcriptase: – Uses mRNA as template to synthesize cDNA. – Produces mRNA/cDNA duplex. – mRNA is partially digested with RNAse H: ▪ Produces gaps in RNA strand. – 3′- ends of mRNA serve as primers for DNA Poly I. – Results in double-stranded cDNA molecule—used for cloning. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 15  11/6/2024 Library Screening Library screening: – Used to sort through library and isolate specific genes of interest Probes: – Used to screen library and recover clones of specific gene – A probe is any DNA or RNA sequence complementary to target gene of sequence being identified. – Probe must be labeled or tagged (chemical or color reactions). Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17.3 The Polymerase Chain Reaction Is a Powerful Technique for Copying DNA Copyright © 2021 Pearson Education Ltd. All Rights Reserved 16  11/6/2024 Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR): – Rapid method of DNA cloning – Extends power of recombinant DNA. – Eliminates need to use host cells for cloning. – Copies specific DNA sequence via in vitro reactions: ▪ Amplifies target DNA sequences present in very small quantities in population of molecules. ▪ Sources include dried blood, semen, or hair Copyright © 2021 Pearson Education Ltd. All Rights Reserved PCR Requirements PCR Amplification requires: – Double stranded target DNA – DNA polymerase – Mg2 (cofactor of DNA polymerase) , – Four deoxyribonucleoside triphosphates – Primers: ▪ Short, single-stranded sequences ▪ One complementary to 5′ end and another complementary to 3′ end. Figure 17-7 Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17  11/6/2024 Figure 17-7 Steps in the polymerase chain reaction (PCR). (a) In this schematic representation, a relatively short sequence of DNA is shown being amplified. Notice that the first cycle produces amplified molecules with a strand that extends beyond the target sequence. (b) Repeated cycles of PCR can quickly amplify the target DNA sequence more than a millionfold. Products in part (b) that consist of only the target sequence are outlined and highlighted. Copyright © 2021 Pearson Education Ltd. All Rights Reserved PCR Process Three steps of PCR: – Denaturation—dsDNA is denatured into single strands – Hybridization/Annealing—Primers bind to ssDNA— starting point for DNA polymerase to synthesize new DNA strands – Extension—DNA polymerase synthesizes DNA strands Each cycle results in amplification—products of previous cycle serve as templates for each subsequent cycle (chain reaction). Copyright © 2021 Pearson Education Ltd. All Rights Reserved 18  11/6/2024 Limitations of PCR Limitation of PCR: – Information about nucleotide sequence of target DNA is required to synthesize primer. – Minor contamination from other sources can cause problems (e.g., skin cells from researcher). – PCR cannot amplify long segments of DNA. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Applications of PCR Applications of PCR: – Most widely used technique in genetics and molecular biology – Rapid technique—takes a few hours – Used in genetic testing, forensics, and molecular pathology Copyright © 2021 Pearson Education Ltd. All Rights Reserved 19  11/6/2024 RT-PCR and qPCR Reverse transcription PCR (RT-PCR): – Methodology for studying gene expression (mRNA production by cells or tissues) – Reverse transcriptase is used to generate cDNA. Quantitative real-time PCR (qPCR): – Real-time PCR allows researchers to quantify amplification reactions as they occur in real time. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17.4 Molecular Techniques for Analyzing DNA and RNA Copyright © 2021 Pearson Education Ltd. All Rights Reserved 20  11/6/2024 Agarose Gel Electrophoresis Agarose gel electrophoresis: – Method that separates DNA fragments by size – Smallest piece moving farthest through gel – Fragments can be visualized with staining and illuminating with UV. – Figure 17.8 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-8 An agarose gel containing separated DNA fragments stained with a DNA-binding dye (ethidium bromide) and visualized under ultraviolet light. Smaller fragments migrate faster and farther than do larger fragments, resulting in the distribution shown. Molecular techniques involving agarose gel electrophoresis are routinely used in a wide range of applications. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 21  11/6/2024 Southern Blot (1 of 2) Southern blot: – Hybridization between complementary DNA molecules – Used to identify which clones in library contain given DNA sequence – Identifies number of copies of particular sequence or gene present in genome. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Southern Blot (2 of 2) Southern blot: – Involves separation of DNA fragments by gel electrophoresis. – Transfer (blotting) of DNA binding membrane and hybridization of fragments labeled with probe. – Membrane is washed to remove excess probe. – Overlay of X-ray film for autoradiography – Only fragments hybridized to probe are visible. – Figure 17.9 Copyright © 2021 Pearson Education Ltd. All Rights Reserved 22  11/6/2024 Figure 17-9 (a) Agarose gel stained with ethidium bromide to show DNA fragments. (b) Chemiluminescent image of a Southern blot prepared from the gel in part (a). Only those bands containing DNA sequences complementary to the probe show hybridization. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Northern and Western Blotting Northern blot analysis: – Used to study patterns of gene expression (RNA production) by cells and tissues – Both characterizes and quantifies transcriptional activity of genes. Western blot: – Used for analyzing proteins Copyright © 2021 Pearson Education Ltd. All Rights Reserved 23  11/6/2024 Fluorescent in Situ Hybridization (FISH) Fluorescent in situ hybridization (FISH): – Involves hybridizing probe directly to chromosome or RNA without blotting. – Carried out with isolated chromosomes on slide or in situ in tissue sections or entire organisms. – Helpful when embryos are used for various studies in developmental genetics. – FISH can be used to produce special karyotypes. – Figure 17-10 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-10 In situ hybridization of a zebrafish embryo 48 hours after fertilization. The probe used shows expression of atp2a1 mRNA, which encodes a muscle-specific calcium pump, and is visualized as a dark blue stain. Notice that this staining is restricted to muscle cells surrounding the developing spinal cord of the embryo. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 24  11/6/2024 17.5 DNA Sequencing Is the Ultimate Way to Characterize DNA at the Molecular Level Copyright © 2021 Pearson Education Ltd. All Rights Reserved Sanger Sequencing Dideoxynucleotide chain-termination sequencing (Sanger): – Most common method of DNA sequencing – Small amount of dideoxynucleotides are added. – Dideoxynucleotides: ▪ Deoxynucleotide with a hydrogen at 3′ instead of an hydroxyl group ▪ Causes DNA synthesis to terminate (terminated by ddNTP). ▪ Figure 17-11 Copyright © 2021 Pearson Education Ltd. All Rights Reserved 25  11/6/2024 Figure 17-11 (1 of 2) Computer-automated DNA sequencing using the chain-termination (modified Sanger) method. The inset box at upper right illustrates dideoxynucleotide (ddNTP) structure. (1) A primer is annealed to a sequence adjacent to the DNA being sequenced (usually near the multiple cloning site of a cloning vector). A reaction mixture is added to the primer–template combination. This includes DNA polymerase, the four dNTPs, and small molar amounts of ddNTPs labeled with fluorescent dyes. (2) All four ddNTPs are added to the same reaction tube. During primer extension, the polymerase occasionally (randomly) inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain because the ddNTP does not have the OH group needed to attach the next nucleotide. Over the course of the reaction, all possible termination sites will have a ddNTP inserted, and thus all possible lengths of chains are produced. The products of the reaction are added to a single lane on a capillary gel (3), and the bands are read by a detector and imaging system (4) from the newly synthesized strand. In this case, the sequence obtained begins with 5’-CTAGACATG-3’ as seen in the chromatograph in step 4. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-11 (2 of 2) Copyright © 2021 Pearson Education Ltd. All Rights Reserved 26  11/6/2024 Computer-Automated Sanger-Reaction Computer-automated high-throughput DNA sequencing: – Since the early 1990s, DNA sequencing has been through computer-automated Sanger reaction-based technology. ▪ Generates large amounts of sequence DNA. – Enabled rapid progress of Human Genome Project. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Sequencing Technologies Have Progressed Rapidly (1 of 2) Next-generation sequencing (NGS) technologies: – Simultaneous reactions synthesize DNA from tens of thousands of identical strands. – Use fluorescence imaging techniques to detect new strands. – Generate massive amounts of DNA sequence data rapidly and at reduced costs. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 27  11/6/2024 Sequencing Technologies Have Progressed Rapidly (2 of 2) TGS: Third-generation sequencers: – Based on sequencing a single molecule of ssDNA – SMRT—single molecule sequencing in real time: ▪ Attaches single molecule of DNA polymerase anchored to substrate. ▪ Visualizes in real time the syntheses of a strand of DNA by polymerase. ▪ Figure 17.12 – Other techniques are also being constructed. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17.6 Creating Knockout and Transgenic Organisms for Studying Gene Function Copyright © 2021 Pearson Education Ltd. All Rights Reserved 28  11/6/2024 Gene Targeting and Knockout Animal Models Gene targeting: – Target specific allele, locus, or base sequence and learn its function on gene of interest. Gene knockout: – Loss of function mutation – Disrupt or eliminate specific gene/genes and see “what happens.” – Knockout (KO) mice have revolutionized research. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Knockout (KO) Mice Creating knockout mice: – Construct targeting vector—creates segment of DNA for introduction into cell. – Targeting vector then undergoes homologous recombination with gene of interest and renders it nonfunctional. – Target vector has mutated a copy of gene of interest. – Foreign DNA disrupts the reading frame and produces nonfunctional protein (Figure 17-13). Copyright © 2021 Pearson Education Ltd. All Rights Reserved 29  11/6/2024 Figure 17-13 A basic strategy for producing a knockout mouse. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Target Vectors Introduced into Cells Embryonic (ES) stem cells: – Using ES cells, scientists introduce targeting vectors into cells via electroporation. – ES cell takes in targeting vector, and endogenous enzyme recombinase catalyzes homologous recombination. – Recombinant ES cells are injected into mouse embryo. – Results: chimeras (Figure 17-14) Copyright © 2021 Pearson Education Ltd. All Rights Reserved 30  11/6/2024 Figure 17-14 (a) Microinjecting DNA into a fertilized egg to create a knockout (or a transgenic) mouse. A fertilized egg is held by a suction or holding pipette (seen below the egg), and a microinjection needle delivers cloned DNA into the nucleus. (b) On the left is a knockout or null mouse (− > −) for both copies of the leptin (Lep) gene. The mouse on the right is wild type (+ > +) for the Lep gene. Normal copies of the Lep gene produce a peptide hormone called leptin. The Lep knockout mouse weighs almost five times as much as its wild-type sibling. Copyright © 2021 Pearson Education Ltd. All Rights Reserved Transgenic Animals Transgenic animals: Knock-in animals: – Express or overexpress particular gene of interest (transgene). – Vector with transgene undergoes homologous recombination into host genome. – Vector with transgene can also be put into ES cells and injected into embryos. – Allow for study of effects on appearance and function in mice (Figure 17-15). Copyright © 2021 Pearson Education Ltd. All Rights Reserved 31  11/6/2024 Figure 17-15 Examples of transgenic mice. (a) Transgenic mice incorporating the green fluorescent protein gene (gfp), a popular reporter gene, from jellyfish enable scientists to tag particular genes with green fluorescent protein. Thanks to the expression of gfp, which makes the transgenic mice glow green under ultraviolet light, scientists can track activity of the tagged genes, including activity in subsequent generations of mice generated from these transgenics. (b) The mouse on the left is transgenic for a rat growth hormone gene, cloned downstream from a mouse metallothionein promoter. When the transgenic mouse was fed zinc, the metallothionein promoter induced the transcription of the growth hormone gene, stimulating the growth of the transgenic mouse. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 17.7 Genome Editing with CRISPR-Cas Copyright © 2021 Pearson Education Ltd. All Rights Reserved 32  11/6/2024 Genome Editing with CRISPR-Cas Genome editing: – Removing, adding or changing specific DNA sequences in genome of living cells CRISPR-Cas: – Fastest and most efficient approach – Discovered as genome editing tool by scientists studying how bacteria fight viral infections Copyright © 2021 Pearson Education Ltd. All Rights Reserved CRISPR-Cas9 Molecular Mechanism CRISPR-Cas system uses nuclease Cas9: – Cas9: ▪ From bacterium Streptococcus pyogenes ▪ Has two nuclease domains—creates double strand break in DNA. ▪ Only cuts DNA near specific sequence protospacer adjacent motif (PAM) 5′-NGG-3′. ▪ Cas9 requires single guide RNA (sgRNA) for activity and specificity. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 33  11/6/2024 CRISPR-Cas9 (1 of 2) Using CRISPR-Cas9 to disrupt gene function or correct mutation: – Takes advantage of eukaryotic cell’s dsDNA break repair mechanisms: ▪ Nonhomologous end-joining (NHEJ) ▪ Homology-directed repair (HDR) – Cas9 and sgRNA (single guide RNA) are introduced into cells in order to either disrupt gene function or create nonfunctional allele. – Figure 17.16 Copyright © 2021 Pearson Education Ltd. All Rights Reserved Figure 17-16 CRISPR-Cas9 genome editing. (a) An sgRNA guides Cas9 to cleave a target site adjacent to a PAM sequence. The double-stranded DNA break can be repaired by (b) NHEJ, which introduces insertions or deletions (indels), or by (c) HDR, which can make specific edits using an introduced donor template. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 34  11/6/2024 CRISPR-Cas9 (2 of 2) CRISPR-Cas9—more precise genome editing: – HDR can be tricked into using artificial donor template. – Artificial donor template: ▪ Used to make complex substitutions, deletions or additions ▪ Donor template carries desired edits with sequences that match genomic target. ▪ Target sequence in genome is replaced by sequence on donor template. ▪ Figure 17.16 Copyright © 2021 Pearson Education Ltd. All Rights Reserved CRISPR-Cas Infidelity CRISPR-Cas does have limitations: – Cas9 can cut at off-target sites in genome. – sgRNA may have more than one perfect match. – sgRNA may direct Cas9 to sequences with one or few mismatches. – Solutions in progress: ▪ Modify Cas9 to improve specificity. ▪ Improve sgRNA design via algorithms. ▪ Find alternative enzymes from bacteria or archaea. Copyright © 2021 Pearson Education Ltd. All Rights Reserved 35  11/6/2024 Diverse Applications of CRISPR-Cas CRISPR-Cas9 being used for: – Creation of tomatoes that ripen quickly. – “Bringing back” the woolly mammoth (not yet). – Fight off diseases in livestock. – Modify food crops for additional nutritional traits, pest, and drought resistance. – Most anticipated applications: ▪ Gene therapy ▪ Clinical trials already underway for cancer Copyright © 2021 Pearson Education Ltd. All Rights Reserved 36