Genome engineering using CRISPR/Cas9: what could go wrong? Genome - - PowerPoint PPT Presentation

Genome engineering using CRISPR/Cas9: what could go wrong?

Genome Editing 2020 St Edmund Hall, Oxford, UK 12th March 2020 Katharina Boroviak

Introduction to CRISPR/Cas9

Doudna, Charpentier and colleagues apply Cas9 in gene targeting.

Introduction to CRISPR/Cas9

5’ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 3’ 5’ 3’

genomic target PAM

NNNNNNNNNNNNNNNNNNNN G G U A A C C G G U U A A A C G G U U A A G U U A A UAAGGCUAGUCCGUUAUCAACUU A A A A A A A G G G U C G G U U A A C C G G U UUUUUU A A G G G G U U C UGAA

guide RNA Cas9

NNNNNNNNNNNNNNNNNNNN G G U A A C C G G U U A A A C G G U U A A G U U A A UAAGGCUAGUCCGUUAUCAACUU A A A A A A A G G G U C G G U U A A C C G G U UUUUUU A A G G G G U U C UGAA

guide RNA Cas9

Introduction to CRISPR/Cas9

5’ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 3’ 5’ 3’

Introduction to CRISPR/Cas9

5’ NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 3’ 5’ 3’

NHEJ DSB is repaired Indels, Small or large deletions, inversions and duplications HDR HR Insertion of small ssDNA oligos  loxP, point mutations, … Seamless insertion of targeting constructs  GFP, lacZ, …

Mashiko D, Young SAM, Muto M, et al (2013) Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev Growth Differ. doi: 10.1111/dgd.12113

ESC vs CRISPR/Cas9

~3 – 6 weeks ~8 – 10 weeks 10 - 11 weeks 4 - ? weeks

Mashiko D, Young SAM, Muto M, et al (2013) Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev Growth Differ. doi: 10.1111/dgd.12113

ESC vs CRISPR/Cas9

~2 – 6 weeks ~6 weeks

Is it really this simple?

~3 – 6 weeks ~8 – 10 weeks 10 - 11 weeks 4 - ? weeks

Delivery of CRISPR/Cas9 via cytoplasmic injection

Delivery of Cas9 (mRNA or protein) and gRNA via cytoplasmic injection into one cell mouse embryos (= E0.5) followed by embryo transfer 1-2 hours after injection or culture to blastocyst (= E3.5) for further analysis.

Brendan Doe

Mosaicism

For initial studies (e.g. test different versions of Cas9 mRNA/protein) we targeted tyrosinase (Tyr)

• re-arrangements/indels in this region should be non-lethal
• ability to score mutation events phenotypically (C57BL6/N zygotes: heterozygous mutation = black coat

colour; homozygous mutation = albino patches or complete albinism).

Mosaicism

 Up to 5 different alleles observed in mice and at blastocyst stage

Mouse genome engineering

Routine:

• Critical exon (CE) deletions
• Single nucleotide polymorphism (SNPs)
• Conditional Knock-outs
• Knock-ins (reporters, humanisation, conditional point mutations,

Ectopic/over-expressors)

• EUCOMM/KOMP ‘knockout first’ models

R&D using CRISPR/Cas9 in zygotes:

• Optimization of conditions
• Conditional alleles
• Homologous recombination
• Genomic rearrangements

CRISPR/Cas9 in zygotes ESC

Mouse genome engineering

Routine:

• Critical exon (CE) deletions
• Single nucleotide polymorphism (SNPs)
• Conditional Knock-outs
• Knock-ins (reporters, humanisation, conditional point mutations,

Ectopic/over-expressors)

• EUCOMM/KOMP ‘knockout first’ models

R&D using CRISPR/Cas9 in zygotes:

• Optimization of conditions
• Conditional alleles
• Homologous recombination
• Genomic rearrangements

CRISPR/Cas9 in zygotes ESC

Mouse genome engineering

Routine:

• Critical exon (CE) deletions
• Single nucleotide polymorphism (SNPs)
• Conditional Knock-outs
• Knock-ins (reporters, humanisation, conditional point mutations,

Ectopic/over-expressors)

• EUCOMM/KOMP ‘knockout first’ models

R&D using CRISPR/Cas9 in zygotes:

• Optimization of conditions
• Conditional alleles
• Homologous recombination
• Genomic rearrangements

CRISPR/Cas9 in zygotes ESC

Routine:

• Critical exon (CE) deletions
• Single nucleotide polymorphism (SNPs)
• Conditional Knock-outs
• Knock-ins (reporters, humanisation, conditional point mutations,

Ectopic/over-expressors)

• EUCOMM/KOMP ‘knockout first models

R&D using CRISPR/Cas9 in zygotes:

• Optimization of conditions
• Conditional alleles
• Homologous recombination
• Genomic rearrangements

CRISPR/Cas9 in zygotes ESC

Mouse genome engineering

Genomic Rearrangements

Genomic rearrangements often present in inherited disease and cancer and often involve gross alterations of chromosomes:

• Deletions
• Duplications
• Insertions
• Inversions
• Translocations

Modelling these structural variants in mice required multistep processes in ES cells  Time consuming and limited their availability.

Genomic Rearrangements

Genomic Rearrangements

What are the limits of using CRISPR/Cas9 to generate rearrangements directly in mouse zygotes?

Genomic Rearrangements

Genomic Rearrangements

Genomic Rearrangements

Genomic Rearrangements

Genomic Rearrangements

Injections and births G0 mice with Deletion size Embryos transferred Pups born (%) Deletion (%) Inversion (%) Duplication (%) 155kb 105 46 (44%) 11 (24%) 14 (30%) 1 (2%) 545kb 114 68 (60%) 12 (18%) 12 (18%) 1 (1%) 1.15Mb 103 48 (47%) 14 (29%) 10 (21%) 0 (0%)

Genomic Rearrangements

Genomic Rearrangements

Genomic Rearrangements

Example #1 Founder endpoint PCR: WT, DEL, INV RV Offspring endpoint PCR: WT, DEL, INV RV

X

Genomic Rearrangements

Example #1 Founder endpoint PCR: WT, DEL, INV RV Offspring endpoint PCR: WT, DEL, INV RV

X

Genomic Rearrangements

Example #2 Founder endpoint PCR: WT, DUP1 Offspring endpoint PCR: WT, DUP1

Genomic Rearrangements

Example #2 Founder endpoint PCR: WT, DUP1 Offspring endpoint PCR: WT, DUP1

Genomic Rearrangements

Other examples 1.15Mb region endpoint PCR: INV RV Duplication of yellow and red probes

Genomic Rearrangements

Other examples Chr1 region spanning 664kb from Cfhr1 to Cfh * endpoint PCR: WT

* Unpublished data: Daniel Gitterman, Matthew Pickering

Genomic Rearrangements

Although CRISPR/Cas9 is very efficient in generating genomic rearrangements, be careful on how you characterize them!

• Loss of primer binding sites can result in incomplete genotype.
• Endpoint PCR can identify correct deletion event but the excised fragment can re-integrate locally and potentially

restore activity of the excised gene.

• WT animals used as littermate controls might actually harbour a rearrangement.
• Even if all the PCRs match up, internal fragments can be lost.

Observed similar events in other genomic regions as well as in some cases of our routine critical exon deletions.  Thorough analysis of alleles generated by CRISPR/Cas9 is crucial.  Do not breed F0s together and assume they generate the desired line.

Off Targets?

• Used two gRNAs within exon2 of Tyr locus
• Analyse the genomes of both parents and offspring using WGS
• Separate de novo mutations from variations within the

population/strain

• If off-targets are an issue in our conditions, we would expect to

see an increase of de novo mutations in treated animals compared to the controls.

CRISPR Sequence Number of nucleotide mismatches Off-target totals 1 2 3 4 Tyr2F Target only 1 4 51 55 Target +1 (DNA bulge) 3 5 75

• 83

Target -1 (RNA bulge) 2 2 8 245

• 257

395 Tyr2R Target only 1 2 15 161 178 Target +1 (DNA bulge) 1 1 12 324

• 338

Target -1 (RNA bulge) 4 71 911

• 986

1,502

Off Targets?

experiment

• rigin

experiment #embryos transferred # pups analysed (%) targeting efficiency: number of genotype confirmed pups (% of pups analysed) # with mosaic genotype (%) average variants per embryo historical Cas9 protein (all) 474 142 (30%) 76 (54%) 35 (25%) 1 historical Tyr2R RNP (equiv. conditions) 30 13 (43%) 11 (85%) 7 (54%) 2 current parent 8 8 (100%) 0 (0%) 0 (0%) current No Injection 15 11 (73%) 0 (0%) 0 (0%) current Sham Injection 15 11 (73%) 0 (0%) 0 (0%) current Cas9 only 15 6 (40%) 0 (0%) 0 (0%) current Tyr2F 14 8 (57%) 7 (88%) 6 (75%) 2 current Tyr2R 13 11 (85%) 10 (91%) 7 (64%) 2

• 18/20 alleles detected by MiSeq were also detected by WGS
• The two missing alleles were at low frequency (10%) and hence filtered out during WGS analysis due to low read number

Off Targets?

• In our conditions, using cytoplasmic

injection, we see efficient rate of on- target mutation but no introduction of

• ff-targets
• Pick best available gRNA and compare

design using multiple design tools

Off Targets?

How the controls can skew the results:

Off Targets?

How the controls can skew the results:

Conclusions

So what can we reliable achieve using CRISPR/Cas9?

• Generation of indels, CE deletions
• Introduction of SNPs
• Deletions and rearrangements of small and large genomic regions

Future prospect

• Improvement of homologous recombination/loxP introduction

long single stranded DNA 2cell MI PiTCH Targeting Vectors …….

Acknowledgements THANK YOU!!!

Allan Bradley Team 82 Dave Adams Vivek Iyer Brendan Doe Fengtang Yang Ruby Banerjee Beiyuan Fu Genome Engineering Microinjection Genotyping Colony management Project Management Mouse Informatics Group RSF support staff