While crispr originated as a mechanism in prokaryotes to defend against viral attack, moving the system to eukaryotes poses some challenges given the differences between prokaryotic and (larger) eukaryotic genomes. one challenge is that while a prokaryotic chromosome is easily accessible in the cytoplasm, eukaryotic chromosomes are contained within a nucleus. scientists have circumvented this by adding nuclear localization signals to cas9 to ensure it can access the nucleus in eukaryotic cells. another challenge is that crispr-cas9 can sometimes cause unintended consequences elsewhere in the genome. a particular 20-bp target sequence chosen by researchers—and encoded in the sgrna--may appear more than once in the genome. this is especially likely to happen in eukaryotes, which have much larger genomes than prokaryotes. if cas9 cuts those other sequences, known as off-target sequences, there may be unintended edits that affect the experiment. cas9 itself also has modest infidelity, which means that it does not always cut at sequences that exactly match the 20-bp target sequence encoded in the sgrna. other hurdles can arise due to how long the genome-editing machinery is active in individual cells. depending on the method of introducing cas9 and sgrna into cells, they can either be heritable or transiently expressed. supplying the components as transgenes can make them present in later cell generations in a stable manner if they are integrated into the genome. however, injecting the components into the cell as rna or protein means their presence and active cutting will be transient, with later daughter cells eventually no longer containing the components. this is important because gene edits are not made with 100% efficiency, meaning that not all cells that are targeted will actually be edited. if cas9’s presence is transient, some cells may never be cut and edited. however, if cas9 is stable, there is a higher chance of off-target effects. it is important to note that once a eukaryotic cell’s genome has been edited, those changes will be inherited by its daughter cells, even if crispr-cas9 is no longer active. now, consider a scenario in which researchers are trying to make a mouse model of a disease that is suspected to be caused by simultaneous mutations in two genes affecting kidney development. they develop two sgrnas, each specific for one of the target genes, and introduce the sgrnas and cas9 components into early mouse embryos. when analyzing the resulting mice, they found that some mice have new mutations in more than those two genes, an indication that crispr-cas9 performed off-target cuts. what steps could refine the researchers’ experiment to modify only the two target genes? select all that apply. a engineer a version of cas9 that demonstrates higher fidelity to the target sequence. b insert more cas9 and sgrna during the initial introduction of the components. c make stable transgenic lines so the cutting by cas9 is not transient. d screen the rest of the mouse genome to be sure the 20-bp targets do not appear elsewhere in the genome. e inject the plasmids into younger mouse embryos so more cells are modified.