Genome edi3ng with the CRISPR-Cas9 system

Transcription

1 CRISPR-Cas9 Genome Edi3ng Bootcamp AHA Council on Func3onal Genomics and Transla3onal Biology Narrated video link: hfps://youtu.be/h18hmftybnq Genome edi3ng with the CRISPR-Cas9 system Kiran Musunuru, MD, PhD, MPH, FAHA FINANCIAL DISCLOSURES: None UNLABELED/UNAPPROVED USES DISCLOSURES: None

2 Genome edi3ng Facilitates knockout or knock-in of muta3ons by introducing a double-strand break at a desired site in genome Drama3cally increases the efficiency of mutagenesis Can be used in vitro and in vivo

3 Double-strand breaks The cell has two methods to repair double-strand breaks (DSBs) Non-homologous end joining (NHEJ) brings two free ends together and rejoins them, error-prone Homology-directed repair (HDR) uses sister chroma3d/chromosome as a template to replace the area of the break via homologous recombina3on

4 Double-strand breaks Can fool the cell into using an exogenously introduced DNA vector as a repair template Can even use a single-strand DNA oligonucleo3de (ssdna oligo) as a repair template If a vector or ssdna oligo harbors a muta3on, can exploit HDR to stably introduce the muta3on into the genome

5 Double-strand breaks Gupta and Musunuru. J Clin Invest 2014; 124:

6 Genome-edi3ng tools Zinc finger nucleases (ZFNs) Meganucleases TAL effector nucleases (TALENs) Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated 9 (CRISPR-Cas9)

7 The CRISPR-Cas9 system for genome edi3ng (guide RNA) Jinek et al. elife 2013; 2:e00471 Mali et al. Science 2013; 339:823-6 Cong et al. Science 2013; 339:819-23

8 CRISPR-Cas9 Cas9 nuclease remains the same regardless of target DNA Changing nucleo3des in the guide RNA alters the sequence specificity of the CRISPR-Cas9 complex Can make a new guide RNA in the laboratory in one day Can make a large library (genome-wide) at one 3me Mixing Cas9 with more than one guide RNA allows for mul3plexing targe3ng mul3ple sites at once

9 Knocking out genes with CRISPR-Cas9 CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC GACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ 20-bp protospacer / S. pyogenes CACAGTGGTGCTAAACGTGC guide RNA Cas9 protein G GTGTCACCACGTAATGCACG Genera3on of a double-strand break at genomic site??????gagtgtaccacccctagcaaac CTGACTACGTGGTCTAGTGT??????????????????CTCACATGGTGGGGATCGTTTG GACTGATGCACCAGATCACA???????????? Non-homologous end joining CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC CTGACTACGTGGTCTAGTGTCACCACGTAA---ACGCGGAGTGTACCACCCCTAGCAAAC CTGACTACGTGGTCTAGTGTCACCACGT-----ACGCGGAGTGTACCACCCCTAGCAAAC CTGACTACGTGGTCTAGTGTCACCACG CGCGGAGTGTACCACCCCTAGCAAAC wild-type vs. indels/frameshies

10 Knocking in variants with CRISPR-Cas9 CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC GACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ 20-bp protospacer / S. pyogenes CACAGTGGTGCTAAACGTGC guide RNA Cas9 protein G GTGTCACCACGTAATGCACG Genera3on of a double-strand break at genomic site??????gagtgtaccacccctagcaaac CTGACTACGTGGTCTAGTGT??????????????????CTCACATGGTGGGGATCGTTTG GACTGATGCACCAGATCACA???????????? Homology-directed repair using DNA template (double-strand vector, ssdna oligo) CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC CTGACTACGTGGTCTAGTGTCACCACGTAATTCACGCGGAGTGTACCACCCCTAGCAAAC wild-type vs. site-specific mutagenesis

11 Genera3ng knockout mice with CRISPR-Cas9 Cas9 mrna, guide RNA (grna) AAAA mutagenesis embryo transfer zygote injec3on blastocyst PCR screening 3 weeks to make knockout mice up to 100% efficiency knockout mice

12 Disease modeling in stem cells with CRISPR-Cas9 genome edi3ng wild-type hpsc or ipsc with muta3on mutant hpsc or corrected ipsc ipsc = induced pluripotent stem cells differen3a3on hpsc = human pluripotent stem cells (ipsc or human embryonic stem cells) phenotypic comparisons

13 On-target vs. off-target effects CRISPR-Cas9 can be very efficient as much as 100% mutagenesis in some applica3ons Along with on-target mutagenesis, there can be offtarget mutagenesis elsewhere in the genome Off-target effects thought to be most likely to happen at sites with sequence similarity to on-target site

14 CRISPR-Cas9 systems Streptococcus pyogenes Cas9 - the standard Cas9 used for virtually all research applica3ons to date - well-characterized on-target, off-target effects Staphylococcus aureus Cas9 - smaller than S. pyogenes Cas9, can be packaged in AAV for in vivo delivery - similar on-target efficiency, possibly less off-target effects (Ran et al., Nature 2015)

15 Introduc3on of Cas9 and guide RNA into cells Ligate two short oligos into plasmid encoding guide RNA = pguide Streptococcus pyogenes U6 promoter G(N) 20 guide RNA Fixed plasmid encoding Cas9 and green fluorescent protein (GFP) = pcas9_gfp CAG promoter Cas9 (from S. pyogenes) 2A GFP Plasmids available from Addgene; other S. pyogenes CRISPR-Cas9 plasmids are available

16 Introduc3on of Cas9 and guide RNA into cells Ligate two short oligos into plasmid encoding guide RNA = psaguide Staphylococcus aureus U6 promoter G(N) 21 guide RNA Fixed plasmid encoding Cas9 and green fluorescent protein (GFP) = psacas9_gfp CAG promoter Cas9 (from S. aureus) 2A GFP Plasmids available from Addgene; other S. aureus CRISPR-Cas9 plasmids are available

17 Targe3ng a site in the genome CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC GACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ 20-bp protospacer / S. pyogenes CACAGTGGTGCTAAACGTGC guide RNA Cas9 protein G GTGTCACCACGTAATGCACG Rules for S. pyogenes CRISPR guide RNA design: 1. The protospacer is 20 basepairs in length 2. The protospacer must be posi3oned just before a 3-bp element matching the sequence NGG (N = any nucleo3de) called the protospacer-adjacent mo3f (PAM) 3. The 5 por3on of the guide RNA matches the protospacer sequence, so that it can bind to the complementary strand of DNA 4. To be expressed efficiently from the U6 promoter, the guide RNA sequence must begin with a G 5. Thus, the guide RNA should start with G followed by the 20-bp protospacer = G(N) 20

18 Targe3ng a site in the genome CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC GACTGATGCACCAGAT GCCTCACATGGTGGGGATCGTTTG \ 20-bp protospacer / S. pyogenes CACAGTGGTGCTAAACGTGC guide RNA Cas9 protein G GTGTCACCACGTAATGCACG Picking a target site: break 1. The double-strand break occurs 3 basepairs upstream of the PAM 2. Muta3ons tend to occur right at the site of the double-strand break 3. Cas9 guide RNA complexes can bind on either DNA strand, so check both the forward and reverse strands for possible protospacers/pams 4. Try to pick a guide RNA sequence that will posi3on the double-strand break as close as possible to the site of the desired muta3on (whether an indel or a knock-in variant) 5. Oeen there will be several reasonable choices 6. If possible, avoid GC-rich protospacers (increased chance of off-target effects)

19 Targe3ng a site in the genome CTGACTACGTGGTCTAGTGTCACCACGTAATGCACGCGGAGTGTACCACCCCTAGCAAAC GACTGATGCACCAGA GCCTCACATGGTGGGGATCGTTTG \ 21-bp protospacer / S. aureus TCACAGTGGTGCTAAACGTGC guide RNA Cas9 protein G AGTGTCACCACGTAATGCACG break Rules for S. aureus CRISPR guide RNA design: 1. Similar to rules for S. pyogenes CRISPR design, but 2. The protospacer is 21 basepairs in length 3. The protospacer should be posi3oned just before a 5-bp element matching the PAM sequence NNGRR (N = any nucleo3de, R = G or A), ideally NNGRRT 4. The 5 por3on of the guide RNA matches the protospacer sequence, so that it can bind to the complementary strand of DNA 5. To be expressed efficiently from the U6 promoter, the guide RNA sequence must begin with a G 6. Thus, the guide RNA should start with G followed by the 21-bp protospacer = G(N) 21

20 Tips for iden3fying target site If trying to knock out a gene, pick a target site in the first coding exon (or, if the gene appears to have mul3ple alterna3ve transcripts in UCSC Genome Browser, pick the earliest coding exon that is shared by all transcripts) If trying to knock in a variant, the site selec3on will be constrained should be within bp of the desired variant, and ideally less

21 Tips for iden3fying target site Use cdna sequence (con3nuous coding sequence) to determine the target site and the consequence of a muta3on can download from GenBank Use genome sequence to iden3fy op3mal protospacer/pam sites can download from UCSC Genome Browser If cdna sequence is used to find protospacer/pam sites, will not account for intronic sequences may not successfully target the genome

22 Example Familial combined hypolipidemia Loss-of-func3on muta3ons in the ANGPTL3 gene S17X muta3on (Ser17Ter) is the most commonly reported Task: design S. pyogenes guide RNAs to target the site of the muta3on, both for knockout of the ANGPTL3 gene as well as to knock in the specific muta3on

23 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... S17X muta3on = TCC! TGA No NGG sequences (PAMs) near the muta3on site Mul3ple CCN sequences (reverse PAMs)

24 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... PAM #1: protospacer = ATTCTGGAGGAAATAACTAG Double-strand break occurs 10 bp away from muta3on site, protospacer overlaps muta3on site (reducing recleavage of site, once muta3on is introduced)

25 Reducing re-cleavage by CRISPR-Cas9 Overlap of protospacer and/or PAM with the muta3on site results in a mismatch(es) aeer knock-in One or two protospacer mismatches should reduce but may not eliminate re-cleavage Protospacer mismatches close to PAM will generally reduce re-cleavage more than those away from PAM Disrup3on of PAM (no longer NGG) should eliminate re-cleavage

26 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... PAM #2: protospacer = ATTGTCTTGATCAATTCTGG Double-strand break occurs 1 bp away from muta3on site, protospacer overlaps muta3on site (reducing recleavage of site, once muta3on is introduced)

27 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... PAM #3: protospacer = TGAATTGTCTTGATCAATTC Double-strand break occurs 4 bp away from muta3on site, PAM overlaps muta3on site (reducing re-cleavage of site, once muta3on is introduced)

28 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... PAM #2: protospacer = ATTGTCTTGATCAATTCTGG Best choice on paper, but ideally all three candidates should be tested for DSB ac3vity in a human cell line (e.g., HEK 293 cells) to iden3fy the best guide RNA

29 Example PAM #2: protospacer = ATTGTCTTGATCAATTCTGG To design oligonucleo3des to insert into pguide: First, add G to the beginning of the protospacer to allow transcrip3on by U6 promoter: GATTGTCTTGATCAATTCTGG This is the sequence that needs to be incorporated at the 5 end of the guide RNA

30 Example GATTGTCTTGATCAATTCTGG To design oligonucleo3des to insert into pguide: Second, use the following templates: 5 -CACC-GNNNNNNNNNNNNNNNNNNNN-3 3 -CNNNNNNNNNNNNNNNNNNNN-CAAA-5 These two oligos can be annealed together to form a small insert that can be ligated into pguide

31 Example GATTGTCTTGATCAATTCTGG Templates: 5 -CACC-GNNNNNNNNNNNNNNNNNNNN-3 3 -CNNNNNNNNNNNNNNNNNNNN-CAAA-5 Oligonucleo3des (can buy these): 5 -CACCGATTGTCTTGATCAATTCTGG-3 5 -AAACCCAGAATTGATCAAGACAATC-3

32 Example Upon crea3on of the pguide vector with the guide RNA targe3ng ANGPTL3 S17X site, can be used for: - knockout: since the site is so close to the beginning of the gene, any indel will truncate the protein - knock-in of muta3on: if used with a single-strand DNA oligonucleo3de as a repair template, HDR will allow for knock-in at a low frequency; however, NHEJ will s3ll be ac3ve and result in indels/knockouts at some frequency

33 Example To design single-strand DNA oligonucleo3de for knock-in, start with the mutant nucleo3de(s) and then add flanking genome sequences on either side of the site to serve as homology arms Should use homology arms 40 nucleo3des in length Can use ssdna oligos as long as 200 nucleo3des

34 Example ANGPTL3 coding sequence (from genome sequence) ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTC TAGTTATTTCCTCCAGAATTGATCAAGACAATTCATC ATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTT GCTATGTTAGACGATGTAAAAATTTTAGCCAATG... S17X muta3on = TCC! TGA ssdna oligo (can buy this) = 5 -ATTAAGCTCCTTCTT TTTATTGTTCCTCTAGTTATTTCCTGAAGAATTGATC AAGACAATTCATCATTTGATTCTCTATCTC-3

35 Assessing efficacy The last step is to design PCR primers to amplify the region surrounding the target site (several hundred basepair length is sufficient, as long as the target site is in the center of the amplicon) CEL I endonuclease or T7 endonuclease I (T7EI) assay (detects DNA mismatches within a PCR product) to determine the efficacy of mutagenesis DNA sequencing to iden3fy specific muta3ons