Getting started with CRISPR: a review of gene knockout and homology-directed repair
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The document provides an overview of gene knockout and homology-directed repair using CRISPR. It discusses designing guide RNAs and comparing delivery methods like lipofection, electroporation, and microinjection. It also covers designing repair templates for homology-directed repair to insert or change DNA sequences. Optimization of guide RNAs, delivery method, and repair template design can improve genome editing efficiency.
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Getting started with CRISPR: a review of gene knockout and homology-directed repair
- 1. Getting started with CRISPR: A review of gene knockout and homology-directed repair Justin Barr Product Manager, Functional Genomics Integrated DNA Technologies 1
- 2. Agenda: Getting started with CRISPR • Background – Overview of the basic CRISPR workflow • Planning for gene knockout – Designing the guide RNA – Comparing delivery methods • Homology-directed repair (HDR) – Designing repair templates 2
- 3. CRISPR editing DNA repair pathways • Non-homologous end joining (NHEJ) – Disrupt a gene • Homology directed repair (HDR) – Insertion or change sequence specifically Guide RNA Cas9 protein Ribonucleoprotein (RNP) 3
- 4. Implementing CRISPR-Cas9 genome editing 4
- 7. Considerations for CRISPR design tools • Species/cell line(s) tested • Cas9 source • Guide RNA source • Method of delivery • Basis for results ranking – Off-target score – On-target score – Filtering 7
- 8. Tools used in these examples • UCSC Genome Browser – genome.ucsc.edu • CRISPR Design – Zhang Lab, MIT – crispr.mit.edu 8
- 10. 3-step transfection: Alt-R™ CRISPR System 10 + + gRNA complex formation RNP complex formation RNP delivery Step 1 Step 2 Step 3 15 min 10 min 30–60 min 1:1 1:1 Lipofection: 10 nM Electroporation: 1–4 µM Microinjection
- 12. Delivery method comparison Lipofection • No instrument required • Low RNP amount • High-throughput • Inexpensive • Not compatible with all cell types • Risk of toxicity 12 Electroporation • Works with most cell types (primary, iPSC) • High-throughput options available • High RNP amount • Requires instrument purchase • Consumables can be expensive Microinjection • Model organism generation • Pronuclear delivery often possible • Lower RNP amount • Requires experienced technician • Requires special equipment • Can be expensive
- 13. Detailed protocols available online Detailed lipofection and electroporation guides: www.idtdna.com/crispr-cas9 13 User methods:
- 14. Basic workflow Assemble RNP Design gRNAs Lipofection Electroporation (primary cells, iPSCs, etc.) Microinjection Mouse model generation and other research organisms Collect genomic DNA Analyze 14
- 15. Collecting genomic DNA 15 Wash cells Lyse cells Transfer & vortex Heat Dilute & spin down
- 16. Analyzing results: T7 endonuclease I (T7EI) assay 16 1. PCR amplify region targeted by CRISPR 2. Heat and cool PCR products to allow heteroduplex formation 3. Incubate with T7EI enzyme (cleaves DNA at mismatches) 4. Visualize products by electrophoresis
- 18. 3-step transfection: Alt-R™ CRISPR System 18 + + gRNA complex formation RNP complex formation RNP delivery Step 1 Step 2 Step 3 15 min 10 min 30–60 min 1:1 1:1 Lipofection: 10 nM Electroporation: 1–4 µM Microinjection + HDR template Ultramer® Oligonucleotides
- 19. HDR considerations • Desired mutation size should determine template choice – Point mutations and small insertions or tags • Single-stranded oligos (Ultramer® DNA Oligonucleotides) – Standard desalting purification – Total unit length: 200 nt or less, including homology arms of typically 30–60 nt – Large corrections and insertion of new coding material • Longer homology arms (100–500 bases) – HDR donor plasmid – dsDNA (gBlocks® Gene Fragments) • Design HDR template as close to cut site as possible 19
- 20. Homology directed repair—symmetric templates 20 Flanking arm length 92 72 57 47 37 27 Cas9 cleavage crRNA guide • EcoRI restriction enzyme site (6 bases) inserted into EMX1 • 10 nM Alt-R™ CRISPR RNP transfected into HEK-293 cells via lipofection • Single stranded HDR oligos: standard desalt Ultramer® Oligos – 3 nM dsDNA or ssDNA HDR template co-transfected with RNP • Isolated genomic DNA 48 hr post lipofection – PCR amplified gDNA with PCR primers designed outside the flanking arms of the HDR template sequence – Digested PCR product with T7EI to determine total editing, and EcoRI to determine insertion
- 21. 21 HDR efficiency is optimal with 30–60 nt homology arms T7EI total editing EcoRI insertion Insertion into EMX1 92 nt Homology arm length 27 nt Cleavage(%)
- 22. dsDNA templates integrate by both NHEJ and HDR 22 Single-stranded HDR template Double-stranded HDR template HDR products HDR products NHEJ products DSB or HDR HDR NHEJ ssDNA HDR template dsDNA HDR template or
- 23. HDR efficiency varies with integration site 23 BCKDK-AS-1079 HIF1A-S-210 KHK-S-224 EGFR-S-123379 LDHA-S-2494 EcoRI insertion 57 nt Homology arm length 37 nt EcoRIcleavage (%)
- 24. Designing the HDR repair template 24 WT sequence TATTCTAAGGCGTTACGCTGATGAATATTCTACGGAATTGCCATAGGCGTTGAACGCTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATAAGATTCCGCAATGCGACTACTTATAAGATGCCTTAACGGTATCCGCAACTTGCGATG TATTCTAAGGCGTTACGCTGATGAATATTAAACGGAATTGCCATAGGCGTTGAACGCTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATAAGATTCCGCAATGCGACTACTTATAATTTGCCTTAACGGTATCCGCAACTTGCGATG Desired edit (stop codon) HDR template TATTCTAAGGCGTTACGCTGATGAATATTAAACGGAATTGCCATAGGCGTTGAACGCTAC 30–60 base arm 30–60 base armmutation TTACGCTGATGAATATTCTA TTACGCTGATGAATATTCTA xx
- 25. Designing the HDR repair template 25 WT sequence TATTCTAAGGCGTTACGCTGATGAATATTCTACGGAATTGCCATAGGCGTTGAACGCTAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ATAAGATTCCGCAATGCGACTACTTATAAGATGCCTTAACGGTATCCGCAACTTGCGATG TATTCTAAGGCGTTACGCTGATGAATA TTCTACGGAATTGCCATAGGCGTTGAACGCTAC ||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||| ATAAGATTCCGCAATGCGACTACTTAT AAGATGCCTTAACGGTATCCGCAACTTGCGATG Desired edit (stop codon) HDR template TATTCTAAGGCGTTACGCTGATGAATA TTAAACGGAATTGCCATAGGCGTTGAACGCTAC 30–60 base arm 30–60 base arm TTACGCTGATGAATATTCTA TTACGCTGATGAATATTCTA xx 33 bp insert 33 nt insert
- 26. Synthesis options for HDR templates • Standard DNA oligonucleotides – Up to 100 bases in length • Ultramer® Oligonucleotides – Up to 200 bases in length • Coming soon: Very long, single-stranded DNA fragments – 200–2000 bases – Currently in limited beta testing phase – Interested? Sign up at: http://go.idtdna.com/ssDNA_update.html 26
- 27. Summary • CRISPR guide RNA design has become fast and easy with the availability of many free online tools. • It is important to understand design tool rankings and recommendations, and how these relate to your experiment. • Efficient ribonucleoprotein delivery can be achieved through lipofection, electroporation, and microinjection. Optimized protocols are available for many applications. • Homology-directed repair allows for simple mutations and small insertions. Oligonucleotide repair templates can be easily designed and synthesized for your experiment. 27
- 28. Thanks to the scientists who contributed to these studies 28 – Mark Behlke, CSO – Nicole Bode – Michael Christodoulou – Michael Collingwood – Joe Dobosy – Ashley Jacobi – Sarah Jacobi – Kim Lennox – Jessica Lister – Garrett Rettig – Mollie Schubert – Bernice Thommandru – Rolf Turk – Chris Vakulskas
- 29. Additional resources and support • Protocols, methods, and FAQs – www.idtdna.com/CRISPR-Cas9: Visit the “Support” tab • IDT Scientific Applications Support – appsupport@idtdna.com or visit www.idtdna.com/contactus 29
- 30. THANK YOU 30