Table 2.

Outline of mouse model generation using CRISPR–Cas9

Week	Milestone	Tasks	NHEJ-based indel knockout model	HDR-based knock in model
0–1	Model design	Identify the gene and the region to be targeted	TARGET THE first exon shared by all mRNA isoforms aiming for generating frameshift mutation	Identify the precise region to introduce point mutation, loxP site, or transgene
		Design and validate genotyping strategies	Conventional PCR should to be validated	Conventional or long range PCR and at times Southern blot strategies should be incorporated into design and validated
		Sequence analysis in the region of interest and check for polymorphisms for strain of interest	When working with a strain of mice other than the reference strain, it is essential to have the region of interest sequenced, to identify potential polymorphisms, which could cripple guide RNA recognition and reduce HDR efficiency
		Design sgRNA(s) using software screening against genome of interest	Examine off-target profile and select optimal guide RNA sequences; i.e., likelihood of a frameshift mutation, avoiding sequence with significant off-target matches. Consider use of paired nickase	Examine off-target profile. It is desirable to ensure that upon HDR, target sequence is rendered refractory to further modifications by CRISPR–Cas9. Consider use of paired nickase
		Order reagents for sgRNA synthesis and donor assembly	N/A	Design ssODN for HDR, centered around point mutation, Tag, loxP, or region to be modified. ssODNs for HDR should be of full length PAGE purified
1–2 (KO) 1–3 (KI with ssODN) 1–8 (KI with dsDNA plasmid)	Reagent Preparation	sgRNA and Cas9 mRNA synthesis and quality control	Cas9 mRNA and sgRNA can be synthesized using commercial kits. Cas9 mRNA or protein can also be purchased from vendors. The quality of the RNA samples needs to be confirmed by gel electrophoresis, particularly poly adenylation product
		Donor provision	N/A	Homology arm lengths between 200 nt to 10 kb, unique in sequence and isogenic to the strain of interest. Donor plasmid can be assembled by molecular techniques or synthesized in its entity
2–12 (KO) 3–13 (KI with ssODN) 8–18 (KI with dsDNA plasmid)	Founder Generation	Inject CRISPR–Cas reagents into mouse zygote	Microinjection materials must to be free of protein, chemical carryovers, and of particulate matter
			Cas9 mRNA or protein, sgRNA	Cas9 mRNA or protein, sgRNA and donor ssODN or plasmid
		Implant manipulated zygotes into pseudo pregnant animals	Develop to term. Founder mice may carry homozygous mutations and must be monitored closely for phenotypes
		Identify founder mice at 2–3 weeks of age	Screen DNA isolated from tail biopsy for NHEJ or HDR events by PCR and sequencing. For transgene KI, use Long Range PCR and Southern blot for genotyping Note, mosaicism is very often seen in founder animals and they must be bred to determine actual germline event
12–22 (KO) 13–23 (KI with ssODN) 18–28 (KI with dsDNA plasmid)	Germline transmission	Set up breeding of putative founders	Breed with WT mice of chosen strain to generate F1 s. Crossing between founders is not recommended, as founder may be mosaic, each carry a unique mutant sequence(s) and carry with it unique off-target profiles
		Identify modified F1 offspring	Identify and sequence the event to fully characterize the nature of the allele, e.g., frame shift mutation, large deletion, or correct HDR event

The table outlines the tasks with approximate time lines required for genome editing in mouse