Mouse Genome Editing Using the CRISPR/Cas System

Abstract

The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models.

Keywords: CRISPR/Cas9; gene editing; mutant mouse; pronuclear and cytoplasmic injection; sgRNA.

Figure 1. Overview of CRISPR/Cas mediated mouse genome editing steps

Figure 2. CRISPR design output files

(A) Screenshot of the CRISPER target search results window that shows two clickable icons. (B) Screenshot of the “Guides and off-targets window”. 1) Graphical display of various targets (the guide with the cursor on it gets highlighted in blue). 2) Serial number of guide sequence 3) Ranking of the guides displayed from highest to lowest. 4) Guide sequences (20 bases long) 5) PAM sequence. 6) Off-target sequences for a cursor selected guide (guide #4 on the left in this example) 7) Ranking of the off-targets displayed from highest to lowest. 8) Number of mismatches, 9) Positions of mismatches. (C) Screenshot of double nickase design. 10) Serial number of double nickase pairs, 11) the nucleotide position of pairs with respect to the input sequence 12) Ranking of double nickase pairs displayed from highest to lowest. 13) Graphical display of double nickase pairs targets (the pair with the cursor on it becomes highlighted in green)

Figure 3. Diagram illustrating the Golden Gate Cloning to build sgRNA expression vectors

(A) Type IIs restriction enzymes are used to produce user-defined overhangs. (B) Forward and Reverse oligonucleotides corresponding to the CRISPR target site 5’N(20) are designed adding Esp3I overhangs compatible with those included in the MLM3636 vector. (C) A one-pot digestion-ligation reaction is performed with circular vector and annealed oligonucleotides to obtain the desired sgRNA vector. (D) Colony PCR of MLM3636-based sgRNA vectors. A forward primer mapping on the hU6 promoter sequence and the sgRNA Reverse oligonucleotide (previously used for the annealing reaction) are used for colony-PCR. (E) Correctly assembled plasmids yield a 93bp product: three positive colonies and a negative colony (ntc) are shown.

Figure 4. Representative agarose gel images of CRISPR/Cas RNA components. A and B: sgRNA in vitro transcription

(A) The sgRNA sequence is PCR-amplified from MLM3636-based vector with primers carrying the T7 RNA polymerase promoter on the 5’ extremity. (B) The resulting PCR is used as a template for T7 RNA polymerase transcription in vitro. A typical gel electrophoresis of an sgRNA after T7 RNA polymerase transcription (T7) and after column purification with Nucaway spin columns (Col). C and D: Cas9 in vitro transcription. (C) The Cas9 ORF, including NLS, is PCR-amplified with primers carrying the T7 RNA polymerase promoter on the 5’ extremity. (D) The PCR product is used as a template for in vitro transcription, 5’capping and 3’ polyA-tailing. A representative gel electrophoresis is shown. A supershift is observed after the polyA tailing reaction (pA). Nucaway spin columns are used for RNA purification. (E) A representative gel image of Cas9 mRNA (100 ng), sgRNA (100 ng) and 1:1 injection mix of Cas9 and sgRNA (50 ng each) samples.

Figure 5. Examples of genotyping

(A) A representative T7E1 assay of a wild type and a mutant sample. The T7E1 cleavage products in mutant sample (+) are marked with asterisks. (B) A 689bp fragment centered on the CRISPR binding site is PCR amplified, cloned and sequenced. Sequence alignments and chromatogram showing a 9bp deletion in two independent clones. (C) PCR of mutation insertion site in an oligonucleotide based HDR knock-in experiment showing showing wild type band (arrow) along with higher sized bands resulting from insertion of the oligonucleotides included in the injection mix. A smaller sized band in sample 9 indicates deletion of a few nucleotides. Unequal intensities of the higher/lower sized bands with that of wild type band in some samples indicate mosaicism. (D) An example of internal + external primers PCR showing amplification of the expected band only in the positive samples (# 2 and 5).

Figure 6. An example of Surveyor assay combined with sequencing to detect CRISPR/Cas mutations on X-chromosome

(A) Surveyor assay of DNA samples isolated from CRISPR/Cas injected pronuclei cultured up to blastocysts stage showing negative (sample 4) and positive (remaining) samples. Red arrows indicate cleaved bands of 261 and 184 bp from the 445 bp PCR product. Notably, all samples were positive by sequencing assay including those that were surveyor negative. Note that occasionally surveyor assay result in very weak cleavage products (e.g., sample 2). (B) Direct sequencing of PCR products from wild type, a surveyor positive (sample 3) and a surveyor negative (4) sample. Sample number 4 had deletion of 8 nucleotides even though surveyor assay was negative. This could be due to the sample being male which will have only one X-chromosome. The sample 3 showed typical overlapping peaks after the cut site (arrow) indicative of two (or more; if mosaic) templates: this sample could be a female and either only one allele is mutated and/or CRISPR/Cas activity is mosaic.