WORKSHOP: CRISPR in insects (Christine Merlin, Texas A&M University)

Considerations for single guide RNA design Egg microinjections Mutation detection strategies

Generate a DSB at a specific-site within the genome Targeted genome editing ZFNs/TALENs/CRISPR Generate a DSB at a specific-site within the genome Induced DSB Desired or random site-specific modification Cell’s endogenous DNA repair machinery KNOCK-IN KNOCKOUT Homologous recombination Non-homologous end joining Sister chromatid Donor DNA (plasmid) Perfect repair Imperfect repair Deletions Insertions Frame-shifts and truncated proteins

The CRISPR/Cas9 revolution: Programmable RNA-guided DNA endonucleases Emerged as a potentially facile and efficient alternative to ZFNs and TALENs for gene targeting in 2013. In only a few years, more than 1100 papers have been published using CRISPR for gene targeting (55 in insects). CRISPR-mediated gene targeting relies on: - A target-specific gRNA sequence tethered to a tracrRNA - A Cas9 protein or Cas9 encoding mRNA - Target sequence complementary to the gRNA, which should be followed by a Protospacer Adjacent Motif (PAM)

Designing your single guide RNAs  Choosing an appropriate target sequence in the genomic DNA  20 nucleotides followed by the appropriate Protospacer Adjacent Motif (PAM): - For Cas9 to successfully bind to DNA, the target sequence in the genomic DNA must be complementary to the gRNA sequence and must be immediately followed by the correct protospacer adjacent motif or PAM sequence. - The PAM sequence is present in the DNA target sequence but not in the gRNA sequence. The Protospacer Adjacent Motif (PAM) Sequence is essential for cleavage

Designing your single guide RNAs TIP: Pay attention to the origin of the Cas9 used, as the PAM sequence differs as a function of the species When using SpCas9, potential target sites are both (5’-20nt-NGG) and (5’-CCN-20nt)

Designing your single guide RNAs Softwares available online

Designing your single guide RNAs Ex.

Germline targeting by egg microinjection at a which embryonic development stage? Bombyx mori Anterior Yolk Germline precursors Posterior Syncitial preblastoderm

Germline targeting by egg microinjection at a which embryonic development stage? Bombyx mori Monarch butterfly Anterior Anterior Micropyle: Location of sperm entry Yolk Yolk Pronucleus male Pronucleus female Zygote nucleus Germline precursors Posterior Posterior Syncitial preblastoderm “One nucleus” stage

Which strategies to detect mutants? Many options are available but the choice may depend on the model organism Knock-out Knock-in  Restriction site  Simple PCR  T7 Endonuclease I assay  Cas9-based cleavage assay  High Resolution Melt Analysis  Fluorescent reporter tag  Restriction site

Summary of targeting and screening strategies Egg injection strategy Selection strategy ZFN mRNAs Cas9 mRNAs + guide RNA AAAAAAAA TALEN mRNAs AAAAAAAA Micropyle Selection of highly targeted mosaics for backcross Co-inject one nucleus stage embryos (20min AEL) Fertilized egg gDNA Assay of somatic mosaicism in individual larvae Assay lesions at targeted site in larva

Detection of CRISPR-mediated mutagenic lesions using gRNAs with restriction enzyme cut site Uncut Lesions Restriction enzyme cut site …but you may want to use the most efficient sgRNA regardless of the presence of an RE cut site

Detection of CRISPR-mediated mutagenic lesions using a T7E1 in vitro cleavage assay T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA 1. 2. 3. 4. Requirements: No SNP present in the amplicon - Work with isogenic lines - Knowledge of SNP at locus of interest in the population Potential problems: - Not quantitative - T7E1 enzyme is not absolutely mismatch specific; it also nicks dsDNA slowly. Cleaves heteroduplexes

Detection of CRISPR-mediated mutagenic lesions using a T7E1 in vitro cleavage assay What I personally do not like: qualitative but not quantitative (does not allow to select mosaics with high levels of targeting).

Detection of CRISPR-mediated mutagenic lesions using a Cas9-based in vitro cleavage assay See Markert et al, 2016 for details on the assay

Detection of CRISPR-mediated mutagenic lesions using High Resolution Melt Analysis (qPCR) HRMA measures differences in the melt curves between amplified fragments. No SNP allowed The differences may be small depending on the sequence change. Specialized software may be required to detect mutated samples. Less labor intensive than Cas9-based assay but may be less appropriate for non-model species as SNPs should be avoided.

Figure 1. 100 bp amplicons are optimal for HRM detection of nuclease induced small indels. Thomas HR, Percival SM, Yoder BK, Parant JM (2014) High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis. PLoS ONE 9(12): e114632. doi:10.1371/journal.pone.0114632 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0114632

Detection of CRISPR-mediated mutagenic lesions generated using multiple sgRNAs 2 or more sgRNAs 2 or more sgRNAs can be used Genomic deletions can be screened by PCR Potential problem: Increase of off target effects Potential problem: Increase of off target effects Chen et al, Scientific Reports, 2014

Detection of CRISPR-mediated mutagenic lesions using a simple PCR-based protocol Yu et al, 2014, PLoS ONE 9(6): e98282.

Detection of CRISPR-mediated mutagenic lesions using a simple PCR-based protocol Once line established Yu et al, 2014, PLoS ONE 9(6): e98282.

Detection of CRISPR-mediated mutagenic lesions by knock-in of a stop codon cassette containing a restriction site Advantage: Provide control over ORF truncation. Screen by: PCR using - 1 primer specific to the cassette - 1 primer on genomic DNA OR Using a restriction assay Gagnon et al, PLOS One, 2014

Detection of CRISPR-mediated mutagenic lesions using the restriction site present in the KI cassette KI Uncut No KI Lesions KI control Mosaics KI events

Beyond knockouts and knock-ins Use of the Cas9 endonuclease as a DNA-binding domain to activate or repress genes Activation by tethering the VP64 transcriptional activator

Beyond knockouts and knock-ins Use of the Cas9 endonuclease as a DNA-binding domain to activate or repress genes - Repression by occupancy of RNA PolII site using a catalytically inactive Cas9

Vectors to be found on Addgene

ZFNs and TALENs : Chimeric restriction endonucleases engineered to cleave at a specific-site  Function as a pair  Each monomer is composed of: - DNA-binding domain - Cleavage domain (FokI Nuclease)  Spacers requirement different: - 5-7 bp for ZFNs - 12-21 bp for TALENs  Targeted site can be chosen to span an endogenous restriction (in the spacer)

cleaves heteroduplexes Assays to screen mutations Genotyping for the presence of lesions using a T7 Endonuclease I assay. Principle:  T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. PCR your target (ex. mix of WT and mutated products) and proceed to denaturation/re-annealing before subjecting your products to T7 endonuclease I. - 200ng DNA with 2ul 10X NEB2 Buffer (vf=19ul) 95C 5minutes denaturation 95C to 85C (-2C/sec) 85C to 25C (-0.1C/sec) annealing - Add 1ul T7 Endonuclease I (15min at 37C) - Bead purify and run on gel T7 Endonuclease I cleaves heteroduplexes