Article: Editing out five Serpina1 paralogs to create a mouse model...

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Article: Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema
Please write a report on the attached paper, about 5 pages double-spaced, in which you summarize the
1) background and scientific problem addressed, and its significance;
2) the methods,
3) approaches and results; and
4) the conclusions and
5) future directions.
The paper has additional supplemental material, which you should also download from the website and read, and refer to in your paper as appropriate.

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Methods:
Authors have utilized the CRISPER gene editing tool to knock out the conserved regions in exon 2 that is common in all the five paralogues of AAT in mice. These conserved sites were identified such that they have low off-target activity. For this selection Bioconductor software package CRISPRseek was utilized. Briefly, the authors constructed guide RNAs (gRNAs) targeting these conserved regions and microinjected them along with Cas9mRNA in zygotes of C57BL6 mice strain. These zygotes were then implanted in 13 pseudo pregnant female mice and the progeny were screened for the absence of AAT in the serum of weaned out pups. For this direct ELISA was employed using a polyclonal antibody against AAT. Out of a total of 45 pups, authors observed three successful knockouts, two of which were males (#7 and #24) and one was female#31). These were then backcrossed to their parental strains to generate founder lines in which F1 generations were all heterozygotes. The success of knockout generation was further confirmed when adult #7 (male) was crossed with adult #31 (female), and the progeny were all found to be devoid of AAT expression. Western blotting was also performed according to the standard procedure for detection of AAT in the...

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