Getting started with CRISPR: A review of gene knockout and homology-directed repair

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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 Guide RNA Ribonucleoprotein (RNP) DNA repair pathways Non-homologous end joining (NHEJ) Cas9 protein Disrupt a gene Homology directed repair (HDR) Insertion or change sequence specifically 3

4 Implementing CRISPR-Cas9 genome editing 4

5 Basic workflow Lipofection Design grnas Assemble RNP Electroporation Collect genomic DNA Microinjection Analyze 5

6 Gene disruption/knockout 6

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

9 Basic workflow Lipofection Design grnas Assemble RNP Electroporation Collect genomic DNA Microinjection Analyze 9

10 3-step transfection: Alt-R CRISPR System 1:1 Step 1 + grna complex formation 15 min 1:1 Step 2 + RNP complex formation 10 min RNP delivery Step 3 Lipofection: 10 nm Electroporation: 1 4 µm Microinjection min 10

11 Basic workflow Lipofection Design grnas Assemble RNP Electroporation Collect genomic DNA Microinjection Analyze 11

12 Delivery method comparison Lipofection No instrument required Low RNP amount High-throughput Inexpensive Not compatible with all cell types Risk of toxicity 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 12

13 Detailed protocols available online User methods: Detailed lipofection and electroporation guides: 13

14 Basic workflow Lipofection Design grnas Assemble RNP Electroporation (primary cells, ipscs, etc.) Microinjection Mouse model generation and other research organisms Collect genomic DNA Analyze 14

15 Collecting genomic DNA Wash cells Lyse cells Transfer & vortex Heat Dilute & spin down 15

16 Analyzing results: T7 endonuclease I (T7EI) assay 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 16

17 Homology-directed repair (HDR) 17

18 3-step transfection: Alt-R CRISPR System 1:1 Step 1 + grna complex formation 15 min 1:1 Step 2 + RNP complex formation 10 min Step 3 RNP delivery Lipofection: 10 nm Electroporation: 1 4 µm Microinjection + HDR template Ultramer Oligonucleotides min 18

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 nt Large corrections and insertion of new coding material Longer homology arms ( 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 Flanking arm length 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 20

21 HDR efficiency is optimal with nt homology arms Cleavage (%) T7EI total editing EcoRI insertion Insertion into EMX1 Homology arm length 92 nt 27 nt 21

22 dsdna templates integrate by both NHEJ and HDR DSB Single-stranded HDR template HDR products ssdna HDR template HDR products or dsdna HDR template HDR Double-stranded HDR template or HDR NHEJ NHEJ products 22

23 HDR efficiency varies with integration site BCKDK-AS-1079 HIF1A-S-210 KHK-S-224 EGFR-S LDHA-S-2494 EcoRI cleavage (%) EcoRI insertion Homology arm length 57 nt 37 nt 23

24 Designing the HDR repair template WT sequence TTACGCTGATGAATATTCTA TATTCTAAGGCGTTACGCTGATGAATATTCTACGGAATTGCCATAGGCGTTGAACGCTAC ATAAGATTCCGCAATGCGACTACTTATAAGATGCCTTAACGGTATCCGCAACTTGCGATG Desired edit (stop codon) TATTCTAAGGCGTTACGCTGATGAATATTAAACGGAATTGCCATAGGCGTTGAACGCTAC ATAAGATTCCGCAATGCGACTACTTATAATTTGCCTTAACGGTATCCGCAACTTGCGATG HDR template TTACGCTGATGAATATTCTA xx TATTCTAAGGCGTTACGCTGATGAATATTAAACGGAATTGCCATAGGCGTTGAACGCTAC base arm base arm mutation 24

25 Designing the HDR repair template WT sequence TTACGCTGATGAATATTCTA TATTCTAAGGCGTTACGCTGATGAATATTCTACGGAATTGCCATAGGCGTTGAACGCTAC ATAAGATTCCGCAATGCGACTACTTATAAGATGCCTTAACGGTATCCGCAACTTGCGATG Desired edit (stop codon) TATTCTAAGGCGTTACGCTGATGAATA TTCTACGGAATTGCCATAGGCGTTGAACGCTAC 33 bp insert ATAAGATTCCGCAATGCGACTACTTAT AAGATGCCTTAACGGTATCCGCAACTTGCGATG TTACGCTGATGAATATTCTA xx TTAAACGGAATTGCCATAGGCGTTGAACGCTAC HDR template TATTCTAAGGCGTTACGCTGATGAATA 33 nt insert base arm base arm 25

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 bases Currently in limited beta testing phase Interested? Sign up at: 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 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 28

29 Additional resources and support Protocols, methods, and FAQs Visit the Support tab IDT Scientific Applications Support or visit 29

30 THANK YOU 30