$($B$5)$ In $2018$ the first $($and to our knowledge the only$)$ humans $($twins$)$ with their genomes modified by CRISPR$/$Cas$9$ were reported to be mutants in the CCR$5$ gene. This gene encodes a receptor protein that is the target of the HIV virus, so the goal was to make the twins resistant to infection, in common with humans homozygous for naturally occurring mutations in the gene.

$40($B$5$a$)$ What reagents would have been introduced into fertilized egg to generate these mutants $($assuming the goal was to generate just any type of loss of function mutation$)?$ One would be Cas$9,$ what would the other be$?$

A: bacteriophage RNA B: crRNA

C: tracrRNA

D: guide RNA

$41($B$5$b$)$ How is a loss of function mutation generated by this procedure using just these reagents?

A: Cas$9$ cleavage in the region homologous to the RNA directly generates a deletion to generate a frame shift B: Cas$9$ cleave in the region homologous to the RNA and then automatically inserts one or two bases to generate a frame shift

C: After cleavage by Cas$9$ in the region homologous to the RNA, the double strand break is repaired by non$-$ homologous end joining which occasionally makes a mistake inserting or deleting some bases that will often result in a frame shift

D: After cleavage by Cas$9$ in the region homologous to the RNA, the double strand break is repaired by homology directed repair which occasionally makes a mistake inserting or deleting some bases that will often result in a frame shift.

$42($B$5$c$)$ A natural human mutation $($in individuals resistant to HIV infection$)$ corresponds to a $32$ bp deletion in CCR$5$ that introduces a premature stop codon. The investigator trying to make the CCR$5$ mutations in the twins wanted to generate this specific natural mutation in them $($i$.$e$.$ wanted to replace the sequence of the twins wild$-$type CCR$5$ gene with the sequence from the naturally occurring mutant gene$)$; was the approach modified from just using the reagents in $($b$)?$

A: No$,$ some of the mutations generated would be expected to produce this specific mutation quite readily B: Yes, you could use a different RNA that corresponds to the region deleted

C: Yes, you need to use a different nuclease that makes two double strand breaks in DNA positioned $32$ bp apart

D: Yes, you also need to introduce DNA that corresponds to the genomic region but includes the $32$ bp deletion so that the DSB produced by Cas$9$ can now be repaired by homology directed repair using that DNA

$43($B$5$d$)$ The experiment was partially successful, resulting in mutant copies of the receptor in the twins $($and a $3$ year jail sentence for the investigator$).$ Are these mutant twins the first human transgenics?

A: Yes

B: No these aren't transgenics because only the endogenous CCR$5$ gene was modified C: No$,$ other transgenic humans have been produced already