A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

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\'Jany of the molecular and catalytic properties of aldolase A, the classical muscle enzyme, and aldolase B, liver aldolase, have been defined.' Although these enzymes have generally similar molecular properties, they have different primary structures and can also be readily distinguished by their catalytic properties.2 In this paper, we report the existence of a third fructose diphosphate aldolase termed "aldolase C" which is distinct from aldolases A and B. These three parent forms of aldolase appear to be structurally homologous since hybrids of these enzymes can be formed by dissociation and reassociation of mixtures of the parent aldolases. Five forms, including three hybrids, are detectable by zone electrophoresis and by chromatography for each two-membered set of parent aldolases. Hybrid forms of the aldolase A-B and aldolase A-C sets are found in extracts of tissues which are known to contain aldolases A and B or A and C, respectively. The fructose diphosphate aldolase activity of all tissues examined can be related to the presence of the parent FDP a-dolases A, B, C, or their hybrids. The number of hybrids found (three) in each two-membered aldolase set is difficult to reconcile with a three-chain model of the enzyme, but it is easily harmonized with a molecule composed of four subunits. In spite of considerable chemical and physical evidence supporting the three-chain model, we have been led to consider the latter possibility seriously.Materials and Methods.-Enzymes and reagents: Rabbit muscle aldolase A (Boehringer) had a specific activity ranging from 10 to 12 units/mg protein. Aldolase B, prepared by the method of Rajkumar, Woodfin, and Rutter,3 had a specific activity of 1.5 units/mg protein. Glyceraldehyde-3-phosphate dehydrogenase was purchased from Boehringer. NaFDP, NAD, and NADH were procured from Sigma Chemical Co. Nitroblue tetrazolium and phenazine methosulfate were obtained from Aldrich Chemical Co.Assays: Aldolase activity was assayed as described elsewhere.4 A unit of enzymatic activity is defined as the cleavage of 1 Mmole of substrate (FDP or F1P) per min.Immunochemical analysis: Preparation of antibodies against aldolases A and B and the methods of immunochemical analyses have been described elsewhere.4 5 Preparation of tissue extracts: The tissues were excised, minced, and homogenized in the cold for 15 sec in presence of 2 vol (w/v) of 0.01 M Tris Cl and 0.001 M EDTA, pH 7.5. The homogenate was centrifuged at 100,000 g at 20 for 60 min.Zone electrophoresis: Zone electrophoresis was performed in 0.06 M barbital, 0.01 M jB-mercaptoethanol, pH 8.6, on 63/4' cellulose acetate strips (Gelman) at 250 v for 90 min with the origin equidistant from the electrodes. The strips were stained for aldolase activity by a modification of a procedure developed by Crawford.6 A 0.5% Noble agar solution in 0.01 M NaAsO4, pH 7.5, containing 0.01 M NaFDP (Calbiochem), 0.001 M NAD, 0.12 mg/ml glyceraldehyde-3-phosphate dehydrogenase, 0.024 mg/ml phenazine methosulfate, and 0.4 mg/ml nitroblue tetrazolium ch...

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Endonuclease activity upon depurinated DNA was measured in extracts of cultured fibrobrasis from xeroderma pigmentosum patients. Cell lines from complementation groups A, B, C, E, and the XP-variant -had slightly reduced levels of activity, but cell lines from complementation group D had one-sixth of the normal activity. An Cell lines developed from skin biopsies of these individuals have been reported to be defective in excision repair of DNA damage introduced by ultraviolet light or by various chemical agents (2,3). This defect is inferred from a reduction in the overall rate of DNA repair synthesis, and it remains unclear at which step the repair pathway is affected. A few XP-cell lines have been tested for defects in early steps of excision repair: two were shown to be unable to excise pyrimidine dimers from DNA (4), and another one was reported to be defective in the first step of repair, the endonucleolytic cleavage presumably near UV lesions of the DNA (5). Somatic cell hybridization studies have recently shown that mutations which lead to the XP phenotype fall into five or six complementation groups (6, 7). This number exceeds the number of steps postulated for excision repair, suggesting that the repair pathway is regulated in a very complex manner or that there exist multiple, or branched pathways of repair.Several enzyme activities believed to be involved in excision repair have been measured in extracts of XP-cell lines. Pedrini et al. (8) found no significant differences in the levels of polynucleotide ligase activity, DNA polymerase, single-strand exonuclease and double-strand endonuclease. Bacchetti et al. (9) compared endonucleolytic activity specific for UV-irradiated DNA in normal and two XP-cell lines and detected no difference. Under their assay conditions the major UV-specific endonuclease activity in fibroblast extracts did not cleave the DNA near pyrimidine dimers, but at other undetermined lesions. In an attempt to Abbreviation: XP, xeroderma pigmentosum. identify excision repair endonucleases we have also found that extracts from a normal cell line and from the XP-cell lines CRL 1157 and CRL 1160 (see Materials and Methods) contained equal levels of an endonuclease activity which preferentially cleaves UV-irradiated DNA at sites which are about 100 times less frequent than the number of pyrimidine dimers in the DNA. However, these studies led us also to compare endonuclease activity with depurinated DNA as a substrate in extracts from normal and XP fibroblasts. Such an activity has been previously reported in many microorganisms, plants and mammals (10-13). MATERIALS AND METHODSGrowth of Cells and Preparation of Extracts. Fibroblast cell lines from skin biopsies were obtained from the American Type Culture collection (ATCC). Strain numbers, complementation groups and relevant properties are summarized irk Table 1. Cells were grown at 370 in 32 ounce prescription bottles with 50 ml of Dulbecco's modified Eagle's medium (Gibco), supplemented with 15 mM N-2-hydroxyethylpiperazine...