Using CRISPR for genetic alteration

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1 Using CRISPR for genetic alteration Joffrey Mianné. Mary Lyon Centre, MRC Harwell.

2 CRISPR/Cas origins Origin of the CRISPR/Cas system: Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein RNA-based adaptive immune system found in procaryotes Specific recognition and cleavage of invading nucleic acids Type II CRISPR/Cas system from Streptococcus Pyogenes is one of the simplest crrna tracrrna Cas9 protein

3 CRISPR/Cas origins From bacterial immune system to genome engineering tool: Jinek & al, 2012

4 CRISPR/Cas for genome engineering From bacterial immune system to genome engineering tool: PAM Protospacer Adjacent Motif (-NGG) sgrna Adapted from Ran & al, Cell

5 CRISPR/Cas for genome engineering Nickase system Recommendation: - 5 overhang - Offset between -4 nt to +20 nt Jinek & al, 2012 Ran & al, Cell

6 CRISPR/Cas for genome engineering Adapted from Shan & al, 2014 Non-Homologous End Joining NHEJ Homology Directed Repair HDR Micro-injection: Cas9 mrna sgrna(s) DNA donor template (ssodn or plasmid) Pronuclear injection in 1 cell stage embryos

7 CRISPR/Cas for genome engineering Knock-Out by introduction of an indel Introduction of point mutations (ssodn donor) WT / WT WT / HDR Tailored deletion Introduction of tags / LoxP sites / complex sequences (ssodn [short] or plasmid [long] donor) is proving difficult through direct injection into zygotes

8 CRISPR/Cas for genome engineering Point mutation project design with Cas9: sgrna selection = compromise As close as possible from the point mutation to introduce As specific as possible (low number and low probability of potential off-target sites, especially on the targeted chromosome) Possibility to mutate the PAM (silent mutation) in order to prevent re-processing of correctly mutated allele (if not possible, try to introduce silent mutations within the seed region) ssodn design 60nt homology arms Contain the intended modification + silent mutation(s) Keep the genotyping step in mind while designing

9 CRISPR/Cas for genome engineering Example: 60nt HA 60nt HA Targeted amino acid: 5 -CGG (Arg) 5 -TAG (Stop)

10 CRISPR/Cas for genome engineering CRISPR design tool:

11 CRISPR/Cas for genome engineering Example: 60nt HA 60nt HA Additional silent point mutations G C to kill the PAM sequence of sgrna_#1 (5 -CTG 5 -CTC = Leu) Targeted amino acid: 5 -CGG (Arg) 5 -TAG (Stop) Additional silent point mutation needed within the PAM sequence of the guide used. This additional modification will prevent the system to re-process/recut the allele repair through Homology Directed Repair (HDR) with the donor oligo (= expected allele).

12 Genotype complexity and mosaicism Cas9 mrna sgrna(s) DNA donor template Pronuclear injection in 1 cell stage embryos = Mutagenesis event Mutant heterozygous (2 alleles) Mutant mosaic (3 alleles) Mutant mosaic (5 alleles)

13 Genotype complexity and mosaicism PCR product subcloning: Subcloning Transformation Each colony contains 1 plasmid Mixed PCR products Sequencing of each individual product Each plasmid contains 1 PCR product

14 Genotype complexity and mosaicism Germ line transmission assessment: X Mutant founder (F0) WT F1 population = Heterozygous: WT / To determine Ex: X Allele not transmitted New allele WT : A C T A G T T A C A G Mut: A C T A C A G G G A T Correct mutation

15 Real life example Project design: Introducing a point mutation in FTO CGT GCT (Arg Ala) Sequencing orientation

16 Real life example PCR product subcloning: Subcloning Allele 1 sgrna #4 Allele 2 Reference Allele 1 Allele 2 Allele 3 Targeted mutation Allele 3

17 Real life example Germ line transmission assessment: X Reference Allele 1 Allele 2 Allele 3 sgrna #4 Targeted mutation WT F1 population WT/WT (Allele 3) WT/Correct point mutations (Allele 1) Targeted mutation Breeding and phenotyping

18 Summary CRISPR/Cas9 technology can be used to efficiently introduce indels, tailored deletions and point mutations through direct injection into zygotes The outcome is unpredictable. Quality control of the alleles obtained through CRISPR/Cas9 mutagenesis is essential and complex F0 should be treated as mosaic Phenotype data should be acquired from F1 onwards, once the alleles have been segregated and fully characterised

19 Interesting websites CRISPR design tools Poly Peak Parser: Interesting papers Jinek & al, Science Singh & al, Genetics Yang & al, Cell Yang & al, Nature Protocols. 2014