A CRISPR-Cas9 mediated knock in mice refers to a genetic engineering mouse that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus . Typically, this is done in mice since the technology for this process is more refined and there is a high degree of shared sequence complexity between mice and humans .
A common use of knock in technology is for the creation of disease models. It is a technique by which scientific investigators may study the function of the regulatory machinery (e.g. promoters) that governs the expression of the natural gene being replaced. This is accomplished by observing the new phenotype of the organism in question. Because of the success of gene knock in methods thus far, many clinical applications can be envisioned. Knock in of sections of the human immunoglobulin gene into mice has been shown to allow them to produce humanized antibodies that are therapeutically useful .
There are several different procedures of producing knock in mice; the following is a typical example:
1. The gene to be inserted is isolated from a mouse gene library. Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbour sequence, except that it is changed sufficiently to make the gene inoperable. Usually, the new sequence is also given a marker gene, a gene that produces an observable change (e.g. fluorescence). In addition, a second gene, such as herpes tk+, is also included in the construct in order to accomplish a complete selection.
2. Embryonic stem cells are isolated from a mouse blastocyst (a very young embryo) and grown in vitro. For this example, we will take stem cells from a white mouse.
3. The new sequence from step 1 is introduced into the stem cells from step 2 by electroporation. By the natural process of homologous recombination some of the electroporated stem cells will incorporate the new sequence with the insered gene into their chromosomes in place of the original gene. The chances of a successful recombination event are relatively low, so the majority of altered cells will have the new sequence in only one of the two relevant chromosomes–they are said to be heterozygous.
Cells that were transformed with a vector containing the RFP gene and the herpes tk+ gene are grown in a solution containing Ganciclovir in order to select for the transformations that occurred via homologous recombination. Any insertion of DNA that occurred via random insertion will die because they test positive for both the RFP gene and the herpes tk+ gene, whose gene product reacts with Ganciclovir to produce a deadly toxin. Moreover, cells that do not integrate any of the genetic material test negative for both genes and therefore are discarded as a result of FACS.
Fig 1. Procedure of CRISPR-Cas9 Knock in Mice.
Fig 2. One-step Generation of Mice Mediated by CRISPR-Cas.
4. The embryonic stem cells that incorporated the inserted gene are isolated from the unaltered cells using the marker gene from step 1.
5. The inserted embryonic stem cells from step 4 are inserted into a mouse blastocyst. For this example, we use blastocysts from a grey mouse. The blastocysts now contain two types of stem cells: the original ones (from the grey mouse), and the inserted cells (from the white mouse). These blastocysts are then implanted into the uterus of female mice, where they develop. The newborn mice will therefore be chimeras: some parts of their bodies result from the original stem cells, other parts from the inserted stem cells. Their fur will show patches of white and grey, with white patches derived from the inserted stem cells and grey patches from the recipient blastocyst.
6. Some of the newborn chimera mice will have gonads derived from inserted stem cells, and will therefore produce eggs or sperm containing the inserted gene. When these chimera mice are crossbred with others of the wild type, some of their offspring will have one copy of the inserted gene in all their cells. These mice will be entirely white and are not chimeras, however they are still heterozygous.
7. When these heterozygous offspring are interbred, some of their offspring will inherit the inserted gene from both parents; they carry no functional copy of the original unaltered gene (i.e. they are homozygous for that allele).
One-step generation of mice carrying reporter and conditional alleles by CRISPR-Cas mediated genome engineering (Figure 2).
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4. Yang et al. (2013). One-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering. Cell. 154: 1370-1379.